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Peptidomics-based Approach Reveals the Secretion of the 29-Residue COOH-Terminal Fragment of the Putative Tumor Suppressor Protein DMBT1 from Pancreatic Adenocarcinoma Cell Lines¹

Growth Factor Division [K. Sas., K. Sat., Y. A., K. Yam.] and Central Animal Laboratory [K. Yan.], National Cancer Center Research Institute, Tokyo 104-0045, and Department of Surgery II, Yamaguchi University School of Medicine, Yamaguchi 755-8505 [M. O.], Japan

	ABSTRACT

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Deleted in malignant brain tumors 1 is a putative tumor suppressor protein in brain, lung, esophageal,gastric, and colorectal cancer. Here we report the mass spectrometric identification of a 3335 Da peptide, which was found in serum-free conditioned medium from 5 of 15 pancreatic adenocarcinoma cell lines but not from 35 carcinoma cell lines and 2 nonmalignant pancreatic duct cell lines. The peptide was the 29 COOH-terminal amino acids from deleted in malignant brain tumors 1. It is suggested that the peptide is generated inside the cells by limited proteolysis and extracellularly secreted. Our peptidomics-based approach will help screen candidate marker peptides for a particular type of cancer.

	Introduction

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Peptides that are secreted from a particular type of cancer cells and stable in body fluids may serve as a tumor marker. This notion is supported by the establishment of pro-gastrin-releasing peptide (31–98; proGRP) as a useful tumor marker specific to small cell lung carcinoma (1 , 2) . The discovery process for new peptide markers may be expedited by recent advances in MS³ . SELDI MS is a type of affinity MS that shares a basic idea with the MALDI MS (3) . SELDI permits the differential display of proteins on various crude biological samples (4) ; however, CM has attracted little attention because low concentrations of cell-derived proteins provide an obstacle to the discovery of target proteins, still less peptides. To overcome this issue, we established a single-step sample preparation method for enriching peptides in SFCM amenable to SELDI MS (5 , 6) .

Despite low protein concentrations (10–50 µM levels), SFCM from cultured cells has several advantages in screening secretory polypeptides. First, even established cell lines are shown to secrete tumor markers currently used in clinical practice, as exemplified by the production of the peptide GRP in small cell lung carcinoma lines (7) . Second, SFCM contains no appreciable amount of serum components that would otherwise mask the detection of cell-derived peptides present in much lower abundance. Third, the relative homogeneity of SFCM favors the biochemical purification of a target. Fourth, it is supposed to be rich in stable peptides that can survive a culture condition of 37°C for a given time period.

To screen for potential marker peptides of pancreatic adenocarcinoma, we performed SELDI MS on SFCM from various carcinoma cell lines as well as nonmalignant pancreatic duct cell lines. We found a peak with a mass of 3335 Da in 5 of 15 pancreatic adenocarcinoma cell lines. Two types of tandem mass spectrometric techniques identified the peptide as the COOH-terminal fragment of DMBT1, the putative tumor suppressor protein in malignant brain tumors and some digestive tract cancers (8) . Our data suggest that this peptide is cleaved from the parent DMBT1 protein by limited proteolysis and secreted to CM.

	Materials and Methods

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Cell Lines.
The sources of cell lines were as follows: pancreatic carcinoma (PANC-1, FA-6, QGP-1, Capan-1, BxPC-3, AsPC-1 SUIT-4, PSN-1, YPK-1, YPK-2, PK-1, PK-8, PK-45P, PK-59, JHP-1, KP-1N, KP-2, and H-48N), colorectal carcinoma (HT-29, COLO 201, LS 180, and LoVo), gastric carcinoma (MKN-1, MKN-7, MKN-28, and MKN-74), and mammary carcinoma (MDA-MB-231, MDA-MB-361, SK-BR-3, and T-47D) were described previously (5 , 6) . Colorectal carcinoma (DLD-1, WiDr, HCT-15, and SW480), gastric carcinoma (Kato III, NUGC-4, H-111, SH-10, and HGC-27), cholangiocarcinoma (HuCCT1 and TFK-1) were from the Cell Resource Center for Biomedical Research (Tohoku University, Miyagi, Japan). Prostate carcinoma (LNCaP.FGC and Du 145), mammary carcinoma (MDA-MB-468 and MCF7), choriocarcinoma (JEG-3), embryonal carcinoma (Tera-1), and normal diploid embryonic lung fibroblast (MRC-5) were purchased from American Type Culture Collection (Manassas, VA). Gastric carcinoma (HSC-41 and HSC-43) were described in Ref. 9 . Prostate carcinoma (PC3) was from the Japanese Cancer Research Bank. QGP-1 was provided by Dr. Haruo Iguchi (National Kyushu Cancer Center, Japan). All of the cancer cell lines but PANC-1, QGP-1, FA-6, Du 145, HSC-43, Kato III, NuGC-4, MKN-1, T-47D, JEG-3, HGC-27, and Tera-1 were established from adenocarcinomas. HPDE4 and H6C7 cell lines were cultured as described previously (6) . The synthetic furin inhibitor dec-RVKR-cmk was purchased from Alexis Co Biochemicals (San Diego, CA). Brefeldin A was from Wako Chemicals (Osaka, Japan).

Preparation of SFCM.
Cancer cell lines were grown in RPMI 1640 (Sigma) supplemented with 10% FCS, penicillin, and streptomycin in a 100-mm dish unless otherwise stated. After reaching confluency, SFCM was prepared as described previously (5) . After a 24-h incubation, CM was centrifuged, and supernatant was stored at -20°C.

Sample Preparation and SELDI MS.
CM (750 µl) was diluted with an isovolume of 0.17 N acetic acid and applied on a SCX membrane (Millipore) as described previously (6) . For reproducibility, each sample was added to two separate spots on a SELDI C16 hydrophobic interaction chip (Ciphergen Biosystems, Fremont, CA), and samples from at least more than two different passages were examined. Mass spectra acquisition and peptide differential display were performed as described previously (5) . Bovine insulin was used as an external calibrant, with mass accuracy of a PBS II SELDI mass spectrometer (Ciphergen) being better than 1000 ppm.

Partial Purification of the 3335-Da Peptide for MS/MS.
Capan-1 SFCM was processed through SCX membranes. Disulfide bridges in the SCX eluate were reduced in 50 mM NH4HCO3 and 50 mM DTT at 56°C for 30 min. Cysteine residues were subsequently modified by iodoacetamide at a final concentration of 200 mM at 37°C for 30 min. The sample was separated by gel filtration with a Superdex Peptide PC 3.2/30 column (Pharmacia) on a Smart HPLC system (Pharmacia). Target fractions were located by SELDI MS.

MS/MS for Peptide Identification.
The peptide-rich fraction was divided in half and lyophilized. One aliquot was reconstituted in 5 µl of 0.5% trifluoroacetic acid/50% acetonitrile and added to a SELDI C16 chip. The chip was mounted with 2,5-dihydroxybenzoic acid and analyzed on a mass spectrometer that combines a SELDI ion source with a selection quadrupole (Q), a collision cell (q), and a time-of-flight (TOF) fragment ion analyzer (SELDI QqTOF; Refs. 4 , 10 ). Each peptide of interest underwent collision-induced dissociation to acquire tandem mass spectra. The other aliquot was reconstituted in 20 µl of 25 mM NH4HCO3 containing 2.5 ng of sequencing grade lysyl-C endoproteinase (Roche) and incubated overnight at 37°C. The digest was loaded to an automated µLC ESI MS/MS system, consisting of a HPLC Magic 2002 (Michrom BioResources, Auburn, CA) connected on-line to a LCQ Deca iontrap tandem mass spectrometer (Thermoquest, San Jose, CA). The HPLC system was first equilibrated with 0.1% formic acid-5% ACN, and then a linear gradient of 5–60% ACN was applied >20 min for analysis. Tandem mass spectra were searched against theoretical tandem mass spectra of peptide sequences in the Nr and Swissprot databases by the Ms-Tag program⁴ for SELDI QqTOF, and Sequest (Thermoquest) and Mascot (Matrix Science, London, United Kingdom) for µLC ESI MS/MS, respectively.

Antibodies.
Monoclonal antibody (anti-DMBT1h12), raised against the first NH₂ terminus of DMBT1(26–40), was provided by Dr. Jan Mollenhauer (11) . The antiserum to detect the target COOH-terminal peptide, designated L129, was prepared against a synthetic peptide (LQTPPRREEEPR) corresponding to DMBT1(2415–2426).

IP-SELDI MS.
Culture supernatant (500 µl) was incubated overnight at 4°C with L129 antiserum (1:100) and then with 20 µl of slurry of Protein G Sepharose (Pharmacia) for 30 min. After five washings with saline, the beads were immersed in 10 µl of 0.2% trifluoroacetic acid to release bound material. Four µl of the supernatant were analyzed by SELDI MS. To detect intracellular peptides, cells were rinsed with saline and lysed in IP buffer [0.5% TX-100, 5 mM EDTA, 1 mM PMSF, 20 µM dec-RVKR-cmk, and Tris-HCl (pH 7.5)]. Cleared supernatant was likewise immunoprecipitated.

Immunoblotting.
Immunoblotting was performed as described previously (11) . Anti-DMBT1h12 and L129 antiserum were used at a dilution of 1:2000 and 1:4000, respectively.

	Results

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SELDI MS Profiling of SFCM to Screen for Differentially Expressed Peptides in Pancreatic Adenocarcinoma Cell Lines.
CM was analyzed to profile peptides from 50 carcinoma cell lines, including 15 pancreatic adenocarcinoma, 3 pancreatic nonadenocarcinoma (PANC-1, FA6, and QGP-1), 6 mammary carcinoma, 8 colorectal adenocarcinoma, 11 gastric carcinoma, 3 prostate carcinoma, 2 cholangiocarcinoma, 1 choriocarcinoma, and 1 embryonal carcinoma cell lines, many of which (38 of 50) are derived from adenocarcinoma. The nonmalignant pancreatic duct cell lines H6C7 and HPDE4 (6) , established from normal pancreatic ducts, were used as a control for pancreatic adenocarcinoma. Fig. 1 shows representative SELDI mass spectra in a mass/charge (m/z) range from 2000 to 7000. They were used to locate differentially expressed peptides. A peak was found at m/z 3335 (arrowheads in Fig. 1 ) in 5 (Capan-1, SUIT-4, PK-8, YPK-2, and PSN-1) of 15 pancreatic adenocarcinoma cell lines but not found in the other cell lines examined.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Representative SELDI mass spectra of peptides from cell lines. The peak with m/z 3335, a differentially expressed peptide, was marked by arrowheads.

View larger version (37K): [in this window] [in a new window]   Fig. 2. SELDI QqTOF tandem mass spectrum acquired from the precursor ion with m/z 3334.737.

View larger version (23K): [in this window] [in a new window]   Fig. 3. Antiserum specificity assessed by IP-SELDI MS. A, the antiserum but not preimmune serum from the same rabbit immunoprecipitated the peptide in culture supernatant from normally growing Capan-1 cells. B, immunoblotting with the antiserum recognized a 250-kDa protein in total cell lysate from Capan-1. Twenty µg of total cell lysate were used/lane.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Detection of the 3335-Da peptide by IP-SELDI MS. A, semiquantitative analysis of the peptide in CM from normally growing cells. The right panel shows examples for negative expression. B, Capan-1 cells were exposed to dec-RVKR-cmk for 48 h, which was replenished every 12 h because of its labile property in CM (14) . C, recovery of the 3335-Da peptide from Capan-1 cell lysate. D, Brefeldin A experiments. Capan-1 cells exposed to the drug for 20 h decreased the peptide secretion in a dose-dependent manner. The values indicate a signal to noise ratio ± SE from three independent measurements.

The 3335-Da Peptide Is Intracellularly Generated and Extracellularly Secreted.
The amino acid sequence immediately NH₂-terminal to the DMBT1 cleavage site, RSKR, is reminiscent of the consensus recognition site for the proprotein convertase furin (13) . It is reported that glioma cells release active furin into culture supernatant, thereby releasing bioactive transforming growth factor ß molecules from precursor molecules (14) . This conversion is shown to be inhibited by the furin synthetic inhibitor dec-RVKR-cmk (14) . To examine if our peptide would be cleaved outside cells, culture supernatants from Capan-1 cells treated with the furin inhibitor for 48 h were analyzed. However, extracellular cleavage was negated because the inhibitor did not affect the peptide amount (Fig. 4B) . Next, Capan-1 cell extract was immunoprecipitated with L129 antiserum, in the presence of 5 mM EDTA, 1 mM PMSF, and 20 µM dec-RVKR-cmk to suppress possible in vitro cleavage. The peptide was already present in cell extract (Fig. 4C) , suggesting that it is intracellularly cleaved, presumably by furin-like proprotein convertase(s). Consistent with this, increasing concentrations of Brefeldin A, an inhibitor of translocation of secretory proteins from the endoplasmic reticulum to the Golgi apparatus (15) , lowered the peptide secretion (Fig. 4D) .

	Discussion

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Peptides and small proteins (<1 kDa) are not yet covered by standard proteomics research tools, typically two-dimensional gel electrophoresis in combination with MS (12) . The term "peptidomics," advocated by two independent groups (16 , 17) , refers to alternative strategies to comprehensively analyze peptides in complex biological mixtures in which they are fractionated by liquid chromatography and then identified by MS/MS. In this study, we adopted a different peptidomics approach by using mass spectrometric techniques to identify secretory cancer marker peptides; SELDI MS was used to screen differentially expressed peptides in SFCM, which were subsequently identified with MS/MS techniques.

The COOH-terminal 29-amino acid fragment of DMBT1, with a molecular mass of 3335 Da, was identified as a peptide apparently specific to pancreatic adenocarcinoma cells. Our IP-SELDI studies indicate that the peptide was intracellularly generated before secretion. Thus, we could reveal a hitherto unknown molecular form of DMBT1 in pancreatic adenocarcinomas. Another noteworthy point is that our sample preparation also allowed the acquisition of mass spectra by the conventional MALDI MS for peptide profiling in CM (data not shown). This also means that we can take advantage of the ability of MALDI collision-induced dissociation to identify individual peptides in mixtures (10) . In fact, a number of proteins or peptide fragments known to have a secretory property are being identified.⁵ Taken together, these findings indicate the validity of our approach to screening secretory marker peptides.

The DMBT1 gene is now considered a candidate tumor suppressor gene for brain, lung, esophageal, gastric, and colorectal cancer (8) . Immunohistochemical studies using anti-DMBT1h12 reveal that the skin, colon epithelium, and renal collecting tubule show immunoreactivity (11 , 18) . On the basis of these observations, we suspected that the 3335-Da peptide might be produced by some normal tissues and released into circulation. Thus far, the peptide was not detected by IP-SELDI MS in 2 mm of serum from two normal subjects (purchased from Biowhittaker, Walkersville, MD). The development of a sensitive assay for this peptide could be helpful for further evaluation.

SAGE gives a clue to the abundance of mRNA transcripts. SAGE is supposed to produce a comprehensive profile of gene expression and can be used to search for tumor markers at the mRNA level (19) . The National Cancer Institute SAGE database⁶ lists 113 libraries (as of Feb. 15, 2002) from various sources, which contains four pancreatic cancer cell lines, two primary cultures of normal pancreatic duct cells, and two surgical specimens of pancreatic cancer. DMBT1 mRNA is given one well-characterized SAGE tag (CTTCTCATCT), significant numbers of which are found in the pancreatic cancer cell lines Capan-1, Capan-2, and HS766T but not found in the two libraries from primary duct cells. These SAGE data support our immunoblotting data, especially DMBT1 overexpression in Capan-1. Of note, two libraries from a single patient with gastric adenocarcinoma contain a significant number of DMBT1 tags. Although our 11 gastric carcinoma cell lines did not express the 3335-Da peptide, additional clinical evaluation should also be made on gastric adenocarcinomas.

The DMBT1 gene has a NH₂-terminal signal peptide sequence (8) . Because the secretory nature has been suggested by immunohistochemical studies alone (11 , 18) , we addressed this issue in a straightforward way using Capan-1 cells. However, our attempt to detect the whole DMBT1 protein from 1 ml of Capan-1 CM by IP immunoblotting was unsuccessful, even in the presence of the protease inhibitors EDTA and PMSF to minimize degradation. In contrast, the small 3335-Da peptide was consistently recovered in the same immunoprecipitates after a 4 h-incubation period. In addition, the peptide tended to accumulate in CM up to 72 h, suggesting that it is relatively stable.

The amino acid sequence immediately NH₂-terminal to the identified peptide, RSKR, suggests its cleavage by furin or furin-like processing proteases, which are ubiquitously expressed in all cell types (13) . The occurrence of the peptide in cell extracts strongly suggests that the peptide is intracellularly cleaved from the parent DMBT1 protein. To our knowledge, this is the first demonstration that a defined portion of a candidate tumor suppressor protein is secreted by a particular cell type. Additional studies will be needed to identify the responsible enzyme. Because COOH-terminal peptides cleaved by furin or furin-like proteases are known to function as an extracellular mediator of signaling (13) , the 3335-Da peptide may have any biological role in pancreatic cancer.

Our study points to a new approach to marker discovery. It should be stressed that other techniques currently applied to marker discovery such as the genome-wide analysis of mRNA expression or the conventional proteome analysis (19 , 20) would not lead to the identification of this peptide. Overall, our peptidomics-based approach for the discovery of candidate secretory marker peptides was verified by the identification of the last COOH-terminal 29 amino acids of DMBT1 in pancreatic adenocarcinoma. Although the expression and secretion of this peptide in the CM seemed to be limited to pancreatic adenocarcinoma cell lines thus far, additional studies are needed to demonstrate that the cleavage is specific to these cells. In the upcoming study, we are going to evaluate its occurrence on clinical specimens and sera if it would serve as a tumor marker specific to pancreatic adenocarcinomas. Besides the search for tumor markers, our approach will greatly expedite the discovery of differentially expressed secretory peptides in a particular context.

	ACKNOWLEDGMENTS

 
We thank Dr. Yi Min She (Department of Physics & Astronomy, University of Manitoba, Canada) and Dr. Kenji Saitoh (Ciphergen Biosystems) for MS/MS analysis of the peptide, and Dr. Jan Mollenhauer (Deutsches Krebsforschungszentrum, Heidelberg, Germany) for the monoclonal antibody.

	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 This work was supported in aid from the Ministry of Health, Labor and Welfare (MHLW) in Japan for Cancer Research and the MHLW Second-Term Comprehensive 10-year Strategy for Cancer Control. K. Sat. is a recipient of a Research Resident Fellowship from the Foundation for Promotion of Cancer Research.

² To whom requests for reprints should be addressed, at E-mail: ksasaki{at}ncc.go.jp

³ The abbreviations used are: MS, mass spectrometry; MS/MS, tandem mass spectrometry; SELDI, surface-enhanced laser desorption ionization; MALDI, matrix-assisted laser desorption ionization; CM, conditioned medium; SFCM, serum-free CM; DMBT1, deleted in malignant brain tumors 1; SCX, strong cation exchange; HPLC, high-performance liquid chromatography; µLC, microcapillary liquid chromatography; ESI, electrospray ionization; IP, immunoprecipitation; PMSF, phenylmethylsulfonyl fluoride; SAGE, serial analysis of gene expression.

⁴ Internet address: prospector.ucsf.edu/ucsfhtml4.0u/mstagfd.htm.

⁵ K. Sasaki, manuscript in preparation.

⁶ Internet address: www.ncbi.nlm.nih.gov/SAGE/.

Received 4/ 8/02. Accepted 7/11/02.

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