Nonpolarized Secretion of Human Meprin in Colorectal Cancer Generates an Increased Proteolytic Potential in the Stroma1

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[CANCER RESEARCH 59, 1127–1133, March 1, 1999] Nonpolarized Secretion of Human Meprin ␣ in Colorectal Cancer Generates an Increased Proteolytic Potential in the Stroma1

Daniel Lottaz, Christoph A. Maurer, Dagmar Hahn, Markus W. Bu¨chler, and Erwin E. Sterchi 2 Institute of Biochemistry and Molecular Biology [D. L., D. H., E. E. S.], and Department of Visceral and Transplantation Surgery [C. A. M., M. W. B.], Inselspital, University of Bern, 3000 Bern, Switzerland

ABSTRACT plasmic reticulum, but whereas meprin ␤ is transported to the plasma membrane as a transmembrane protein, meprin ␣ is specifically re- Epithelial cells of the normal human colonic mucosa secrete an astacin- tained in the endoplasmic reticulum to allow the removal of its type metalloprotease, meprin ␣ (E. C. 3.4.24.18, N-benzoyl-L-tyrosyl-p- membrane anchor, leading to its secretion into the extracellular me- aminobenzoic acid hydrolase), into the intestinal lumen. We found that ␣ Caco-2 cells, a colon carcinoma cell line, expressed endogenous meprin ␣, dium (23, 24). However, meprin also may be retained at the plasma ␤ which was secreted at both the basolateral and apical plasma membrane. membrane via dimerization with transmembrane meprin (21). The expression of meprin ␣ in colorectal cancer was confirmed using It has been shown for the mouse (25), as well as for the human Northern blot analysis. On tissue sections, a diversity of carcinoma cells enzyme (26), that meprin ␣ and ␤ are differentially expressed in the with varying immunoreactivity for meprin ␣ was observed. Western blots small and large intestines. Biosynthetic studies using organ-cultured of a series of 11 paired samples of carcinomas and normal control colon human intestinal explants have shown that this leads to different tissue revealed that meprin ␣ protein accumulated at significant levels in proportions of cell surface-bound and secreted meprin ␣. In the small 6 carcinomas at Union International Contre le Cancer tumor stages I–IV. intestine, both meprin ␣ and ␤ are expressed by intestinal epithelial In contrast, the protease was never detected in normal control tissue cells, and meprin ␣/␤ oligomers accumulate at the brush border samples. Meprin ␣ zymogen was activated in the tumor tissue, as shown membrane of enterocytes. In contrast, epithelial cells of the colonic by a 3-fold increase in enzymatic activity. In conclusion, we describe a ␣ cancer-specific sorting of meprin ␣, leading to a redistribution with mucosa only express meprin , which, thus, is secreted into the consecutively increased proteolytic activity in the tumor stroma. Because intestinal lumen. the protease is known to cleave extracellular matrix components in vitro, Meprin ␣ and meprin ␤ are typically sorted to the apical plasma meprin ␣ may contribute to tumor progression by facilitating migration, membrane, as shown by the accumulation at the brush border mem- intravasation, and metastasis of carcinoma cells. brane of enterocytes and the secretion into the intestinal lumen of the colon. Biosynthetic studies with transfected MDCK cells confirm an ␣ ␤ INTRODUCTION apical sorting of meprin and in polarized epithelial cells (21). Here we show that meprin ␣ is endogenously expressed by Caco-2 The involvement of proteases in growth and progression of colo- cells, a human polarized colon carcinoma cell line (27), and that the rectal tumors is well recognized. Protease inhibitors have been proven protease is secreted both from the apical as well as from the basolat- to be efficacious anticancer therapeutics (1–4). The degradation of the eral plasma membrane. We hypothesized that in colorectal tumors in basement membrane and extracellular matrix components is thought vivo, the basolateral secretion of meprin ␣ by colon carcinoma cells to be a prerequisite for tumor invasion and is a hallmark of malig- will expose extracellular matrix components and other stromal ele- nancy (5, 6). Carcinoma cells, as well as mesenchymal and inflam- ments to an increased proteolytic potential in the vicinity of tumor matory cells in the tumor stroma, contribute to an increased proteo- cells. We, therefore, assessed the expression and proteolytic activity lytic activity through the expression of matrix metalloproteases and of this protease in colorectal tumors as compared with normal colon the up-regulation of the plasminogen-activation system (7–12). We tissue. propose a further component, meprin (N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase, E. C. 3.4.24.18; Refs. 13 and 14), to the protease network in colorectal tumors. Human meprin, a metallopro- MATERIALS AND METHODS tease of the astacin family (15, 16), has first been purified from human intestinal epithelial cell membranes due to its activity against the Acquisition of Human Normal Colon Mucosa and Carcinoma Samples. artificial substrate paba-peptide,3 which is used in a noninvasive Collection of tissue material from organ donors and from patients during surgery was approved by the Ethical Committee of the Medical Faculty, exocrine pancreas function test (17). Meprin degrades a broad range University of Bern. of substrates from biologically active peptides to extracellular matrix RT-PCR. Total RNA was extracted from fresh mucosal scrapings of the components in vitro (18, 19). The enzyme is an oligomeric protein small and large intestines of organ donors and from Caco-2 cells using the complex of two similar isoforms, meprin ␣ and meprin ␤ (20, 21), guanidine-isothiocyanate method (28). Reverse transcription was performed on which are encoded at two independent gene loci (22). 100 ng of total RNA, as previously described (29). Human meprin ␣- and The posttranslational processing of meprin ␣ and ␤ is different. ␤-specific sequences (corresponding to nucleotides 1808–2037 and 1449– Both are synthesized as type I transmembrane proteins in the endo- 1883, respectively) were amplified using the following primers at 1 ␮M (Microsynth, Windisch, Switzerland): human meprin ␣-5Ј:5Ј-CGGGATCCA- GACTGAAGTTCCCTCTAAAG-3Ј; human meprin ␣-3Ј:5Ј-GGAAT- Received 7/1/98; accepted 12/31/98. Ј ␤ Ј Ј The costs of publication of this article were defrayed in part by the payment of page TCGGGTCACATGGGTCTCTGAAGTAC-3 ; human meprin -5 :5-CG- charges. This article must therefore be hereby marked advertisement in accordance with GGATCCATTTCCACTTGATCTCTGGAGCC-3Ј; human meprin ␤-3Ј: 18 U.S.C. Section 1734 solely to indicate this fact. 5Ј-GGAATTCAGTGCAGACACCGTCATTTTTAC-3Ј. 1 Supported by Swiss National Science Foundation Grant 32-40571.94 (to E. E. S.) and BamHI and EcoRI restriction sites were included at 5Ј ends of these primers by a fellowship from the “Sonderprogramm akademische Nachwuchsfo¨rderung” by the Swiss Government (to D. L.). to facilitate the subcloning of PCR products. 2 To whom requests for reprints should be addressed, at Institute of Biochemistry and Cell Culture, Biosynthetic Labeling, and Immunoprecipitation. Caco-2 Molecular Biology, University of Bern, Bu¨hlstrasse 28, 3012 Bern, Switzerland. E-mail: cells were cultured in the presence of 20% fetal bovine serum (Sigma Chem- [email protected]. 3 ical Co., St. Louis, MO) in HEPES-buffered (25 mM) MEM containing 4.5 The abbreviations used are: paba-peptide, N-benzoyl-L-tyrosyl-p-aminobenzoic acid; ␮ MDCK, Madin Darby canine kidney; RT-PCR, reverse transcription-PCR; UICC, Union g/liter D-glucose, supplemented with nonessential amino acids (100 M each), International Contre le Cancer. 2mM glutamine, and 100 IE/ml penicillin/100 ␮g/ml streptomycin (culture 1127

medium and all supplements from Life Technologies, Inc., Paisley, United Great Britain) using a peroxidase-coupled antirabbit second antibody (1:10,000 Kingdom). Caco-2 cells were labeled with 50 ␮Ci [35S]methionine/[35S]cys- in 1% dry milk). X-ray films were exposed for 15 min. teine (protein labeling mix; NEN, Boston, MA) at preconfluent and postcon- Meprin ␣ Proteolytic Activity: Paba-peptide Assay. To avoid interfer- fluent time points. To analyze polarized transport of meprin ␣,5ϫ 105 cells ence with the paba-peptide assay, endogenous nonmetalloprotease activity in were seeded onto 25-mm permeable filter supports (0.4 ␮m pore size; Falcon the intestinal tissue was suppressed by the addition of protease inhibitors Becton Dickinson Europe, Meylan Cedex, France) and grown to 10 days after before homogenization (1 ␮g/ml pepstatin, 1 ␮g/ml aprotinin, 5 ␮g/ml leu- confluency. Establishment of a tight monolayer was followed by measuring the peptin, 17.4 ␮g/ml benzamidine, and 1.7 mM phenylmethylsulfonyl fluoride, transepithelial resistance compared with a filter blank (filter support with all inhibitors from Sigma Chemical Co.). Tissue homogenate (100 ␮l, con- culture medium only; Ref. 30). Cells were pulse-labeled for 1 h with 50 ␮Ci taining 120 ␮g of solubilized protein) was mixed with 100 ␮lof40mM [35S]methionine/[35S]cysteine (protein labeling mix, NEN) in methionine-free paba-peptide (Bachem AG, Bubendorf, Switzerland) in 50 mM Tris-HCl, and culture medium and subsequently chased for 24 h with medium containing 10 1mM MgCl2 (pH 7.5), supplemented with 0.5 mM phenylmethylsulfonyl mM cold methionine. Meprin ␣ was immunoprecipitated under denaturing fluoride (Sigma Chemical Co.), and incubated for6hat37°C. Released conditions from cell lysates and from sterile filtered culture media using a benzoic acid was determined using the colorimetric assay according to Bratton-Marshall. After subtraction of absorptions in the presence of 10 mM rabbit antiserum directed against a 300 amino acid NH2-terminal region of human meprin ␣. The antiserum recognizes both human meprin ␣ and ␤ (31). EDTA, paba-peptide hydrolytic activity of homogenates were expressed in Radiolabeled meprin ␣ from cell lysates and culture media was analyzed by IU/g protein. Paba-peptide hydrolysis was inhibited with 10 mM EDTA, but ␮ 7.5% SDS-PAGE under reducing conditions and fluorography. was not affected by 1 M phosphoramidon (Sigma Chemical Co.), which is in ␣ Northern Blot Analysis. 32P-labeled RNA-probes were synthesized from accordance with the inhibitory profile of meprin (18). Background values of 600 ng of linearized plasmids (pBluescript KS-; Stratagene, La Jolla, CA) normal colon mucosa varied from one patient to another. Paba-peptide hydro- containing human meprin ␣ and ␤ 5Ј coding sequences with low similarity lytic activities of colorectal carcinomas were, therefore, related to the normal (nucleotides 1808–2037 and 1449–1883, respectively), using T3 and T7 RNA colon mucosa samples of each patient by calculating the ratio between enzyme polymerases (Boehringer Mannheim, Rotkreuz, Switzerland) in the presence activity in carcinoma tissue and normal colonic tissue. An enriched small of 50 ␮Ci [32P]CTP (NEN). Total RNA (12 ␮g) from normal ileum, normal intestinal brush border membrane preparation was used as a positive control. The statistical significance of the increase in proteolytic activity in meprin colon, and colorectal carcinoma tissue specimens was separated on a denatur- ␣-positive as compared with meprin ␣-negative tumors was confirmed by the ing 1.2% agarose gel containing 1.8 M formaldehyde, blotted onto a nylon two-tailed Wilcoxon rank sum test. membrane (Genescreen; NEN), and hybridized sequentially with 32P-labeled human meprin ␣- and ␤-specific single-stranded RNA probes (32). Samples from pre- and postconfluent Caco-2 cells were probed sequentially with RESULTS randomly labeled full-length cDNAs of meprin ␣ and of ␤-actin. Meprin ␣- and ␤-specific Immunohistochemistry. Meprin ␣- and ␤-spe- Endogenous Expression and Nonpolarized Secretion of Meprin cific rabbit antisera directed against COOH-terminal regions with low simi- ␣ in Differentiated Caco-2 Cells. We found that Caco-2 cells, a ␣ ␤ larity between human meprin and (encompassing amino acids 457–574 differentiated human colon carcinoma cell line, endogenously ex- and amino acids 468–612, respectively) were generated using recombinant pressed meprin ␣ (Fig. 1A). Reverse transcription and PCRs with glutathione-S-transferase fusion proteins, as described elsewhere (26). The meprin ␣-specific primers on total RNA from human small and large specificity of rabbit antisera was confirmed on Western blots, by immunopre- intestines and from Caco-2 cells yielded identical amplimers in all cipitation, and by immunocytochemistry using MDCK wild type and meprin ␤ ␣- or meprin ␤-transfected cells (21, 23). three cases, whereas using meprin -specific primers, only the reac- Dewaxed and rehydrated 2-␮m paraffin sections (on poly L-lysine-coated tion with RNA from the small intestine generated the expected DNA ␣ slides) were pretreated by boiling for 5 min in a microwave oven in 10 mM species. Expression of meprin in Caco-2 cells was further confirmed sodium citrate (pH 6.0) to denature and retrieve antigens and to suppress on the protein level by a Western blot analysis (data not shown). endogenous peroxidase activity. Sections were incubated with rabbit antisera Caco-2 cells differentiate into polarized enterocyte-like cells at or monoclonal antisucrase-isomaltase antibody HBB 3/705/60 (33), biotiny- postconfluency (27, 34). To investigate whether the expression of lated antirabbit or antimouse second antibodies, and an avidin-biotin-peroxi- meprin ␣ depends on the differentation status, we analyzed Caco-2 dase complex (Vectastain ABC-Immunostaining Kit, Vector Laboratories, cells during the growth phase and at postconfluency (Fig. 1, B and C). Burlingame, CA) in 25 mM Tris-HCl, and 140 mM sodium chloride (pH 7.5). Transcription (Fig. 1B) and protein synthesis (Fig. 1C) of meprin ␣ Diaminobenzidine (Immuno Pure Metal Enhanced DAB; Pierce Chemical Co., were up-regulated in postconfluent cell cultures, but were not detect- Rockford, IL) was applied as a chromogenic substrate. Normal rabbit serum able in proliferating cells. The 100,000-Da and 85,000-Da proteins in and a negative control monoclonal antibody (DAKO Diagnostics, Zug, cells correspond to the endoplasmic reticulum-resident precursor and Switzerland) were used as negative controls. ␣ Western Blot Analysis of Tissue Homogenates of Colorectal Carcino- proteolytically processed forms of meprin (23). mas and Normal Colonic Mucosa. Paired samples of colorectal carcinomas The Caco-2 cell line was used as an in vitro model to investigate ␣ and normal control mucosa were taken from 11 patients during elective tumor biosynthesis and secretion of meprin in colon carcinoma cells (Fig. surgery. Using a teflon homogenizer, frozen tissue pieces (weight, 60–200 mg) 2). Pulse-chase experiments were performed in 10-day postconfluent

were disrupted in 5 ml of ice-cold 1.5 mM KH2PO4,8mM Na2HPO4, and 138 cells grown in a transwell culture system with separate basolateral and mM NaCl (pH 7.3), containing protease inhibitors (1 ␮g/ml pepstatin, 1 ␮g/ml apical compartments. Meprin ␣ was immunoprecipitated from cell aprotinin, 5 ␮g/ml leupeptin, 17.4 ␮g/ml benzamidine, and 1.7 mM phenyl- lysates, as well as from apical and basolateral media compartments, methylsulfonyl fluoride, all inhibitors from Sigma Chemical Co.). Homoge- and analyzed by SDS-PAGE and fluorography. After a 24-h chase, the nates were lysed for1hinthepresence of 1% deoxycholic acid and 1% NP40 secreted 95,000-Da form of meprin ␣ was detected in the apical and and adjusted to 1.2 mg of protein/ml (DC Protein assay kit; Bio-Rad Labora- at significant higher amounts in the basolateral compartment. The ␮ tories, Hercules, CA). Solubilized protein/sample (40 g) were separated by 85,000-Da polypeptide in cells corresponds to the COOH-terminally 7.5% SDS-PAGE under reducing conditions and blotted for 1 h onto a trimmed form of meprin ␣ in the endoplasmic reticulum (23). polyvinylidene difluoride-membrane (Millipore, Bedford, MA) using the Human Meprin ␣, but not Meprin ␤, Is Expressed in the Mini-blot system from Bio-Rad. Further incubations were done using 20 mM Tris-HCl and 500 mM NaCl (pH 7.5). Membranes were probed with a com- Normal Colon and in Colorectal Carcinomas. Total RNA from bination of two antisera against COOH-terminal (see immunohistochemistry) normal colon tissue and from colorectal carcinomas were analyzed on ␣ a Northern blot using meprin ␣-specific and meprin ␤-specific RNA and NH2-terminal regions of meprin (Ref. (31); 1:500 and 1:1000, respectively, in 1% dry milk). Meprin-specific bands were visualized using enhanced probes (Fig. 3). Meprin ␣, but not meprin ␤, was expressed in the chemiluminescence (ECL-Kit; Amersham Life Sciences, Buckinghamshire, normal colon (Fig. 3, Lanes 2–6), whereas both were detected in the 1128

No aberrant meprin ␣ transcripts were evident in tumors. As in the normal colon, meprin ␤ mRNA was not detected in the tumor samples (Fig. 3B, Lanes 7–11). Signals for meprin ␣ were much stronger in the ileum as compared with carcinoma and normal colon tissue samples, probably reflecting a higher epithelial cell content and/or higher expression level in the ileum. Using meprin ␣- and meprin ␤-specific immunohistochemistry on tissue sections of colorectal tumors, meprin ␣-positive carcinoma cells were readily identified (Fig. 4A), whereas on consecutive tissue sections, meprin ␤ was not detected in the same tumor (Fig. 4B). A single carcinoma cell typically displayed a uniform staining of the cell body. In a given tumor region, however, a mosaic staining pattern for meprin ␣ was observed, as carcinoma cells with strong immunoreactivity neighbored cells with weak or undetectable signals for meprin ␣. Stromal cells did not exhibit immunoreactivity for meprin. Accumulation of Meprin ␣ in Colorectal Carcinomas, but not in Normal Colon. To this point, we have demonstrated that: (a)a colon carcinoma cell line secretes meprin ␣ from both basolateral and apical plasma membranes in vitro;(b) colorectal carcinomas frequently express meprin ␣ mRNA; and (c) carcinoma cells are pro-

Fig. 2. Nonpolarized secretion of meprin ␣ from Caco-2 cells. Pulse-chase experiment of Caco-2 cells grown in a transwell culture system to 10 days after confluency. After a 24-h chase, radiolabeled meprin ␣ was detected at higher amounts in the basolateral (baso) than the apical (api) compartment.

Fig. 1. Expression of meprin ␣ in Caco-2 cells. A, RT-PCR with human meprin ␣- and human meprin ␤-specific primers on 100 ng of total RNA from normal human colon, small intestine, and Caco-2 cells. Only meprin ␣ mRNA, not meprin ␤, was detected in colon and in Caco-2 cells. No specific bands were observed when reverse transcriptase (RT) was omitted. B, Northern blot analysis of total RNA grown at 50% and 100% confluency and at 2, 6, 10, and 16 days after confluency (Lanes 1–6). Human small intestinal RNA was used as a positive control (Lane 7). The blot was sequentially hybridized with full-length probes for meprin ␣ and for ␤-actin. Ethidium bromide staining of the agarose gel is shown. The amount of the meprin ␣ transcript increases on differentiation of Caco-2 cells. C, correlation of meprin ␣ protein expression with differentiation status of Caco-2 cells. Human meprin was immunoprecipitated from biosynthetically labeled cells and from corresponding culture media before and after confluency, as indicated. No Meprin ␣ was detected at 50% confluency, minor amounts in 100% confluent cells, and increasing amounts at 2–16 days after confluency.

Fig. 3. Northern blot analysis of meprin ␣ and meprin ␤ expression in human normal ileum (Fig. 3, Lane 1), thus confirming the differential expression of colon mucosa and in colorectal carcinomas. Total RNA from normal ileum and colon and from colorectal carcinomas were hybridized with meprin ␣- and ␤-specific RNA probes meprin in the intestine. A single 3.5-kb transcript for meprin ␣ was for human meprin ␣ (A) and human meprin ␤ (B). A single 3.5-kb mRNA for meprin ␣ detected weakly in one tumor sample (Fig. 3A, Lane 7) and at was detected in normal colon mucosa and colorectal carcinomas, whereas meprin ␤ mRNA was not detected. Meprin ␣ 3.5-kb and meprin ␤ 2.6-kb messages are highly significant levels in four tumor samples (Fig. 3A, Lanes 8–11), expressed in the ileum (arrows). Sizes of meprin mRNAs were approximated using 28S indicating that meprin ␣ is frequently expressed in colorectal cancer. RNA and 18S rRNA bands at 4.72 kb and 1.87 kb, respectively. 1129

Fig. 4. Immunohistochemistry for meprin ␣ and meprin ␤ in colorectal cancer. Paraffin sections (2 ␮m) of a colorectal carcinoma were immunostained using a human meprin ␣-specific antiserum (A), a human meprin ␤-specific antiserum (B), and normal rabbit serum (C). Sections shown are from the same tumor region. Some groups of carcinoma cells displayed strong immunoreactivity for meprin ␣ (filled arrowheads), whereas other cells did not exhibit any immunoreaction (open arrowheads). Meprin ␤ was not detected in the same tumor region. No meprin ␣-or␤-positive cells were observed in the tumor stroma. Bar (A–C), 50 ␮m.

duction sites for meprin ␣ in the tumors. We, therefore, hypothesized Table 1 Expression and activity of meprin ␣ compared with sucrase-isomaltase in that the expression and nonpolarized secretion of meprin ␣ by colon colorectal cancer carcinoma cells leads to the accumulation of the protease in the tumor. Patients meprin ␣ Paba-peptide sucrase- (UICC)a (Western blot)b hydrolysisc isomaltased To test this hypothesis, we performed a Western blot analysis of a P1 (III) Ϫ 1.0 Ϫ series of 11 samples of UICC tumor stages I-IV in comparison with P2 (III) ϩ 2.0 Ϫ normal control colon tissue from the same patients (Fig. 5). Multiple P3 (III) Ϫ 1.7 Ϫ ␣ P4 (I) ϩ 3.7 Ϫ forms of meprin , ranging from 75,000–95,000 Da, were detected in Ϫ Ϫ ␣ P5 (IV) 1.0 6 of 11 tumor homogenates. In marked contrast, meprin levels in P6 (II) ϩ 2.5 Ϫ normal colon tissue were always below detection limit. Isolated P7 (II) Ϫ 1.0 Ϫ ϩ Ϫ 70,000-Da bands seen in some samples were not characterized further, P8 (II) 3.0 P9 (I) Ϫ 0e Ϫ but were clearly smaller than known intracellular or secreted forms of P10 (IV) ϩ 2.3 ϩ meprin ␣ and were not associated with a proteolytic activity in these P11 (I) ϩ 4.0 Ϫ tissue samples. We refer to those colorectal carcinomas, which accu- a Tumor stages according to the definition of the UICC are indicated. Meprin ␣-pos- ␣ ␣ itive tumors are indicated in bold. mulate meprin protease, as “meprin -positive.” An association of b Western blot signals are interpreted as positive, if the secreted 95,000-Da form of meprin ␣ protein accumulation with tumor stages (Table 1) was not meprin ␣ was detected. Those tumor samples are defined “meprin ␣-positive.” Correlation observed. coefficient of meprin ␣ protein detection and proteolytic activity: 0.83 (for the calculation, ␣ meprin ␣-positive and -negative tumors were given the value 1 and 0, respectively). Meprin Is Active in Colorectal Cancer. Because meprin is c Proteolytic activity of carcinoma samples against paba-peptide are expressed as ratios synthesized as an inactive zymogen by epithelial cells (23), an accu- between paba-peptide hydrolytic activities of carcinomas and normal control colon mulation of meprin in the tumor does not necessarily implicate an mucosa. d Sucrase-isomaltase expression determined by immunohistochemistry. increased meprin ␣ protease activity. We, therefore, measured meprin e No proteolytic activity was detectable in this carcinoma sample.

␣ activity in tissue homogenates using paba-peptide as substrate (Ref. 18; Fig. 6). In meprin ␣-positive carcinomas, the paba-peptide hydro- lyzing activity was 2.9-fold higher than in the corresponding normal colon mucosa, whereas in meprin ␣-negative carcinomas, this activity was equal to the corresponding normal colon mucosa. Hence, accumulation of meprin ␣ was accompanied by an increased enzyme activity in meprin ␣-positive carcinomas (Table 1). Expression of Human Meprin ␣ in Colorectal Cancer Is Inde- pendent of Sucrase-isomaltase. There are reports on the concomi- ␣ Fig. 5. Accumulation of meprin protease in colorectal carcinomas. Solubilized tant aberrant expression of small intestinal brush border enzymes by protein (40 ␮g) from colorectal carcinomas (c) and corresponding normal colon mucosa specimens (n) from 11 patients were analyzed for meprin ␣ expression on a Western blot. colon carcinoma cells in colorectal tumors, as exemplified by the Patients are designated P1 to P11. Meprin ␣ (75,000–95,000-Da-forms; arrowheads) was prototypic hydrolase sucrase-isomaltase (35). However, we found that ␣ meprin ␣-positive ,ء) detected in carcinoma samples from P2, P4, P6, P8, P10, and P11 carcinomas). In contrast, meprin ␣ was not detected in normal control colon tissue. expression of meprin is not linked to the induction of sucrase- Molecular weight markers in kilodaltons are on the left. isomaltase (Table 1). 1130

which leads to a redistribution of the proteolytic potential of meprin ␣ from the intestinal lumen to the tumor stroma in colorectal cancer. Intestinal epithelial cells transport meprin ␣ apically. This is supported by the accumulation of meprin ␣ together with membrane-bound meprin ␤ at the apical cell membrane of enterocytes in the small intestine and by its secretion from the mucosal surface into the intestinal lumen in the colon (26). In transfected MDCK cells, a polarized kidney epithelial cell line, meprin ␣ is also secreted apically (21). In contrast, differentiated Caco-2 cells target meprin ␣ to the apical as well as the basolateral medium compartment (Fig. 2), distinguishing the colon carcinoma cells from nontransformed epithelial cells. A similar discrepancy has been observed with a recombinant secretory mutant of aminopeptidase N, which is secreted exclusively from the apical plasma Fig. 6. Meprin ␣ proteolytic activity in colorectal carcinomas. Paba-peptide hydrolyz- ing activity was measured in carcinoma samples and related to normal control colon tissue membrane in MDCK cells (36), but from both basolateral and from the same patients. Proteolytic activities in meprin ␣-positive and in meprin ␣-neg- apical plasma membranes in Caco-2 cells (37). ative colorectal carcinomas (six and five patients, respectively) are expressed as ratios to corresponding normal control colon tissues of the same patients. All values in the meprin Like MDCK cells, Caco-2 cells are polarized epithelial cells. Yet, ␣-negative group were below the meprin ␣-positive group. The average increase of the sorting mechanisms for polarized protein transport are different in proteolytic activity was 2.9 in meprin ␣-positive and 1.0 in meprin ␣-negative colorectal these two cell types (30, 38). Sorting of apical proteins occurs directly carcinomas. P Ͻ0.01 (two-tailed Wilcoxon rank sum test). from the trans-Golgi in MDCK cells and seems to depend on sorting signals in the ectodomain (36). Meprin ␣ is secreted via this direct DISCUSSION apical transport route in these cells. In contrast, Caco-2 cells display an additional indirect apical transport route via transcytosis from the We show that the human colon carcinoma cell line Caco-2 on basolateral plasma membrane (39–41). Efficient transcytosis of pro- differentiation endogenously expresses meprin ␣. Using this cell line teins in Caco-2 cells depends on the anchorage to the plasma mem- as an in vitro model to study the sorting of meprin ␣, we demonstrate brane, as shown by the basolateral secretion of an important portion of that an important portion of secreted meprin ␣ is targeted basolater- a secretory mutant of aminopeptidase N (37). In analogy, secreted ally. This observation, in conjunction with an investigation of the meprin ␣, following both the apical and indirect basolateral route in accumulation and enzymatic activity of meprin ␣ in colorectal cancers Caco-2 cells, cannot be scavenged for transcytosis to the apical in vivo, is the basis for the description of a tumor-specific mechanism, domain after the release at the basolateral plasma membrane. There-

Fig. 7. Model of an altered sorting of meprin ␣ in a trans- formed epithelial cell in colorectal cancer. Meprin ␣ is targeted to the intestinal lumen by epithelial cells of the normal colon mucosa (left), where the zymogen is activated by luminal trypsin. A colon carcinoma cell secretes meprin ␣ both to the apical and basolateral side, leading to an accumulation of the zymogen in the stroma (right). The zymogen is activated by trypsin or a trypsin-like protease produced by the carcinoma cell itself or by other cells. The basement membrane, extracellular matrix components, and growth factors are possible substrates for activated meprin ␣.

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1999 American Association for Cancer Research. HUMAN MEPRIN ␣ IN COLORECTAL CANCER fore, the altered sorting mechanism in Caco-2 cells explains the colorectal cancer. We have shown an altered sorting of meprin ␣ in nonpolarized secretion of meprin ␣. colorectal carcinoma cells leading to an aberrant accumulation of We present evidence that similarly to Caco-2 cells, carcinoma cells meprin ␣ in the tumor stroma, thus resulting in a proteolytic potential, in colon tumors also secrete meprin ␣ at the basolateral side. First, which can be activated by proteases from carcinoma cells or from meprin ␣ was evenly distributed at the apical and basolateral domains cells in the tumor stroma. Thus far, meprin has been thought to have of carcinoma cells on immunostainings of tumor tissue sections. a luminal function in the intestine. For the first time, we present direct Second, the analysis of a series of colorectal tumor samples on evidence for a possible tissue-related function of meprin. Western blots revealed a high prevalence of an accumulation of A correlation of meprin ␣ expression with tumor stages was not meprin ␣ protein in tumors. In contrast, meprin ␣ was below detection attempted on the small number of samples investigated However, limit in normal control colon tissue from the same patients, confirm- we find meprin ␣ expressed in tumors of all stages, suggesting a ing its rapid secretion from the normal mucosal surface. Similarly, in potential role of this protease during early, as well as during late, a study on the expression of meprin in mouse intestine, meprin ␣ has phases in tumor progression. To further explore the role of meprin not been detected on Western blots of normal mouse colon, despite ␣ in the progression of colorectal cancer in vivo, additional studies positive mRNA signals on Northern blots (25). In vivo, the basolateral will focus on differences in the prognosis of meprin ␣-positive plasma membrane of colon carcinoma cells faces the tumor stroma. versus meprin ␣-negative colon tumors and on the role of meprin Therefore, meprin ␣, which normally is secreted at the apical plasma during metastasis. membrane and targeted to the intestinal lumen, aberrantly accumu- lates in the stroma in colorectal tumors (Fig. 7). ACKNOWLEDGMENTS Meprin ␣-positive tumors harbored multiple molecular forms of meprin ␣ ranging from 75,000–95,000 Da, yet secreted meprin ␣ in We thank Ursula Luginbu¨hl and Doris Willi for excellent technical assist- Caco-2 cells is a 95,000-Da protein. 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Daniel Lottaz, Christoph A. Maurer, Dagmar Hahn, et al.

Cancer Res 1999;59:1127-1133.

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