Novel protein isoforms of carcinoembryonic antigen are secreted from pancreatic, gastric and colorectal cancer cells
- Keiichi Hatakeyama†1,
- Kanako Wakabayashi-Nakao†1,
- Keiichi Ohshima1,
- Naoki Sakura1,
- Ken Yamaguchi2 and
- Tohru Mochizuki1Email author
© Hatakeyama et al.; licensee BioMed Central Ltd. 2013
Received: 26 July 2013
Accepted: 24 September 2013
Published: 26 September 2013
Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is an oncofetal cell surface glycoprotein. Because of its high expression in cancer cells and secretion into serum, CEA has been widely used as a serum tumor marker. Although other members of CEACAM family were investigated for splice variants/variants-derived protein isoforms, few studies about the variants of CEACAM5 have been reported. In this study, we demonstrated the existence of novel CEACAM5 splice variants and splice variant-derived protein isoforms in gastrointestinal cancer cell lines.
We identified two novel CEACAM5 splice variants in gastrointestinal (pancreatic, gastric, and colorectal) cancer cell lines. One of the variants possessed an alternative minor splice site that allowed generation of GC-AG intron. Furthermore, CEA protein isoforms derived from the novel splice variants were expressed in cancer cell lines and those protein isoforms were secreted into the culture medium. Although CEA protein isoforms always co-existed with the full-length protein, the secretion patterns of these isoforms did not correlate with the expression patterns.
This is the first study to identify the expression of CEA isoforms derived from the novel splice variants processed on the unique splice site. In addition, we also revealed the secretion of those isoforms from gastrointestinal cancer cell lines. Our findings suggested that discrimination between the full-length and identified protein isoforms may improve the clinical utility of CEA as a tumor marker.
Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5; synonyms, CEA, CD66e) is a glycosylated oncofetal antigen and was first found in gastrointestinal cancer tissues [1, 2]. The same research group that initially identified CEA subsequently indicated that CEA levels could be measured in serum from patients with colorectal and other carcinomas , which introduced the possibility of clinical application for diagnosis, monitoring and prognosis. CEA is now generally accepted as a valuable tumor marker for monitoring of several cancers following surgery.
CEA, which belongs to the CEACAM family of the immunoglobulin (Ig) superfamily [4, 5], has been suggested to mediate cell adhesion on tumors [6–8], and the members of this family, i.e., CEACAM1, CEACAM3, CEACAM4, CEACAM6, CEACAM7, and CEACAM8, are also deregulated in various tumors. In this subset, splice variants and protein isoforms were previously identified, and among them, CEACAM1 was found to occur in various alternatively spliced forms . Recently, Tamura et al. revealed that expression of CEACAM1 protein isoforms (CEACAM1-4L and -4S) was associated with cell adhesion in colorectal cancer cells . Despite these findings, relatively few screening approaches for splice variants of CEA have been reported.
In normal colorectal tissues, CEA is actually expressed at the cell surface and released extracellularly. The expression of CEA is probably associated with anoikis as a surveillance mechanism to preserving the normal physiological architecture . The secretion of this molecule that can bind to E. coli and Salmonella expressing type 1 fimbriae [12, 13] is also considered to play an essential role in the host defense mechanism preventing the binding of pathogenic bacteria . However, in cancerous cells, the role of the abundant release of CEA remains unclear.
To determine the expression and secretion patterns of CEA in gastrointestinal tumors, we demonstrated the existence of novel CEA splice variants and splice variant-derived protein isoforms in gastrointestinal cancer cell lines. Furthermore, these isoforms were found to be secreted to the culture medium. These findings suggested that the cancer-released CEA in the blood may include our identified protein isoforms.
Cell cultures and RNA sample preparations
Human gastrointestinal cancer cell lines QGP1, MKN45 and KATO III were purchased from the Japanese Collection of Research Bioresources, HPAC, HPAF II, SNU1 and LoVo from the American Type Culture Collection, and HCA2 and HCA46 from Dainippon Sumitomo Pharma (Osaka, Japan). These cell lines were cultured and maintained in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Invitrogen, Carlsbad, CA), glutamine (0.3 mg/ml), penicillin (100 unit/ml) and streptomycin (0.1 mg/ml) in a humidified 5% CO2 incubator.
After total RNA had been extracted from each pellet using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany), RNA concentration was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA) and total RNA quality was then confirmed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The purified total RNA from cancer cell lines and total RNA derived from normal tissues (Clontech, Mountain View, CA) were then reverse-transcribed using ThermoScript Reverse Transcriptase (Invitrogen) and oligo(dT)20 primers in accordance with the manufacturer’s instructions.
Screening of cDNA for different splice variants of CEACAM5 was performed using the intron-spanning exonic primers listed in Additional file 1: Table S1. PCR amplicons were designed to detect the splice variants and the transcripts registered in GenBank (NM_004363). The synthesized cDNAs were amplified using LA Taq polymerase (Takara Bio, Shiga, Japan) by 35 PCR cycles of 95°C for 30 s, 51°C for 30 s, and 68°C for 60 s.
Rapid amplification of cDNA ends and DNA sequencing analysis
To determine the sequence of novel CEACAM5 transcripts, rapid amplification of cDNA ends (RACE) was conducted using the GeneRacer Kit (Invitrogen) according to the manufacturer’s instructions. The primer sequences are shown in Additional file 1: Table S1. For DNA sequencing analysis, the PCR products were analyzed on 1–2% agarose gels by electrophoresis following by gel staining with SYBR Safe (Invitrogen). The bands visualized under ultraviolet light were isolated and purified using the QIAquick Gel Extraction Kit (Qiagen). The purified samples were then cloned into a pCR 2.1-TOPO vector (Invitrogen) using the TOPO TA Cloning Kit for Sequencing (Invitrogen). Positive transformants were sequenced using the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and an ABI 3130xl Genetic Analyzer (Applied Biosystems). The protein coding sequences obtained by the DNA sequencing have been deposited in DNA Data Bank of Japan (DDBJ), which are accessible through the DDBJ accession number AB852566 and AB852567.
Quantitative real-time PCR
Quantitative real-time PCR (qRT-PCR) was performed using the SYBR Green dye technique and the ABI PRISM 7900HT Fast Real Time PCR System (Applied Biosystems, Foster City, CA). The target fragment was amplified using specific primers (Additional file 1: Table S1) according to the following protocol: preheating at 95°C for 20 s; 40 cycles at 95°C for 1 s and 60°C for 30 s; and dissociation at 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The threshold cycle (Ct) values were converted into absolute copy numbers using a standard curve with purified PCR amplicons that were generated from plasmids containing the sequence of the target transcripts. Nonspecific amplification in qRT-PCR was evaluated using dissociation curves and gel electrophoresis that showed the amplicon size.
Preparation of cell lysate and serum-free conditioned medium
Cells were placed (4 × 106 cells) on 100-mm dishes and incubated until 70%–80% confluence. After incubation, the culture medium was removed and the cells were then harvested with ice-cold PBS. After centrifugation and removal of PBS, cell pellets were treated with lysis buffer containing 7.5 M urea, 2.5 M thiourea, 12.5% glycerol, 50 mM Tris–HCl (pH 7.4), 2.5% N-octylglucoside, 6.25 mM Tris-carboxyethyl phosphine hydrocholine (TCEP), and 1.25 mM protease inhibitor (Sigma-Aldrich, St. Louis, MO), and rotated at 4°C for 1 h. Following the rotation, the samples were centrifuged at 14,000 × g for 30 min at 4°C, and the supernatants were then collected. Protein concentrations were determined by the Bradford assay (BioRad Laboratories, Hercules, CA).
Serum-free conditioned medium was prepared as previously studied . Briefly, cells were placed (8 × 106 cells) on 100-mm dishes, and incubated at 37°C overnight. After removal of the conditioned medium, the cells were washed with PBS (pH 7.4) and then pre-incubated with 8 ml of serum-free medium at 37°C for 1 h. The conditioned medium was then removed and replaced with 3.5 ml of serum-free medium at 37°C for 24 h. The serum-free conditioned medium was collected and centrifuged to remove cell debris. The collected medium was then concentrated using Amicon Ultra Centrifugal Filters (4 mL, 10 K device, Millipore, Bedford, MA) at 4°C and 2,330 × g, and stored at −80°C until further analysis.
Cell lysates were purified using a 2-D Clean-Up Kit (GE Healthcare UK Ltd., Buckinghamshire, England) to precipitate proteins according to the manufacturer’s instructions. The resulting dry pellets were resuspended in 1× Glycoprotein Denaturing Buffer and incubated at 95°C for 10 min. The denatured samples were incubated with a reaction buffer containing 1× G7 Reaction Buffer, 1% NP-40, protease inhibitor, and PNGase F (New England Biolabs, San Leandro, CA) at 37°C for 1 h. The concentrated conditioned medium samples were also denatured and treated with PNGase F as described above. The deglycosylated samples were treated with SDS-PAGE sample buffer solution containing 200 mM DTT.
CEA in the cell lysate or serum-free conditioned medium was detected by immunoblotting with an anti-CEA mouse monoclonal antibody (1:1,000 or 1:3,000 dilution; Clone C6G9, Sigma-Aldrich; this antibody was raised against the CEA isolated from a human colon adenocarcinoma cell line) as the primary antibody, and anti-mouse IgG-horseradish peroxidase (HRP) conjugate (1:3,000 or 1:10,000 dilution; Jackson ImmunoResearch, West Grove, PA) as the secondary antibody. HRP-dependent luminescence was developed using ECL Prime Western Blotting Detection Reagent (GE Healthcare) and detected using a LAS-3000 device (Fuji Film, Tokyo, Japan). To detect actin-ß, which was used as an internal loading control, immunoblot detection was conducted in the same manner described above, except for the use of a mouse monoclonal anti-actin-ß antibody (1:5,000 dilution; Sigma-Aldrich) as the primary antibody.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis
To identify the CEA isoforms, proteins were separated by SDS-PAGE and visualized by silver staining (SilverQuestTM Silver Staining Kit, Invitrogen). Protein bands smearing around 90 kDa, 60 kDa, and 45 kDa were excised manually. Each gel slice was destained with Destainer solution in the staining kit and dehydrated with 100% acetonitrile. After drying under reduced pressure using the SpeedVac (Thermo Fisher Scientific), gel pieces were reduced with 50 μl of 10 mM DTT at 56°C for 45 min and alkylated with 50 μl of 55 mM iodoacetamide for 30 min at room temperature in the dark. The resulting samples were washed and dried using the SpeedVac, and then digested overnight with 8.3 ng/μl trypsin (Promega, Fitchburg, WI) at 37°C. The tryptic digests were purified with ZipTip C18 (Millipore), concentrated using the SpeedVac, and reconstituted in 0.1% formic acid.
LC-MS/MS analysis was conducted on a nanoflow LC-electrospray ionization linear ion trap-time of flight (LC-ESI LIT-TOF) mass spectrometer (NanoFrontier L; Hitachi High-Technologies, Tokyo, Japan). The methods used for each instrument and the strategy for data analysis are were previously described in detail .
Peptide sequences were identified against the SwissProt database (version: 2012_03) with the following parameters: enzyme, trypsin; maximum number of missed cleavages, 1; peptide tolerance, 0.4 Da; MS/MS tolerance, 0.4 Da; fixed modification, carbamidomethylation of cysteine; variable modification, oxidation of methionine and conversion of asparagine to aspartic acid; peptide charge, 1+, 2+, and 3+. The validity of a formerly N-glycosylated peptide was confirmed by the presence of a consensus N-X-S/T (X ≠ P) sequence and deamidation of the asparagine residues.
Identification of novel CEACAM5 exons
Characterization of splice variant 3D
Validation of CEACAM5 mRNA expression levels
Detection of CEA protein isoforms derived from novel splice variants
To detect CEA bands on the theoretical molecular weight calculated from the amino acid sequences, cell lysate samples were treated with PNGase F, which cleaves N-glycan chains from proteins. After PNGase F treatment, bands were clearly detected in three regions (Figure 4B). The theoretical molecular weight was as follows: CEA full-length, 80 kDa; isoform derived from variant 5D, 60 kDa; and isoform derived from variant 3D, 40 kDa. Considering these findings, the band detected in region 1 was considered to represent full-length CEA and the bands in regions 2 and 3 were considered to be the CEA isoforms derived from the novel splice variant 5D or 3D.
Identification of CEA protein isoforms by LC-MS/MS analysis
Peptide sequences identified by LC-MS/MS analysis
Secretion of CEA protein to serum-free conditioned medium
Alternative splicing allows a single gene to generate multiple mRNAs that can be translated into diverse proteins . Many transcripts have been predicted by in silico approaches and registered in public databases (e.g., Ensembl, http://www.ensembl.org) as candidate splice variants [24, 25]. Recently, splice variants and their protein isoforms not registered in these databases were identified by transcriptome analysis [26–28]. Dorard et al. revealed that a novel HSP110 variant in colorectal cancer inhibits the function of wild-type proteins, which results in facilitation of apoptosis and increased chemosensitivity . Thus, it is of importance to discover unpredictable splice variants and protein isoforms, and here, we focused on CEA and its isoforms that were not registered in databases.
The exon structure of many splice variants found in transcriptome analysis was determined using mRNA sequencing and potential GT-AG splice sites. Although we identified two novel splice variants of CEACAM5 (variant 5D and 3D), the splice site between exon 3 and 7 in variant 3D was not defined using this procedure (Figure 2A). To clarify the exon structure of variant 3D, we screened other potential splice sites in the unique sequence shared between exon 3 and 7, which resulted in identification of the specific splice site (AGGC). Because this identified site was present in the shared exons, the GC-AG intron is considered to be generated from pre-mRNA (Figure 2B). These findings raise the possibility that a part of the pre-mRNA derived from CEACAM5 is processed by a U2-type spliceosome. Recently, Peng et al. reported another CEACAM5 variant that does not include a major splice site between exons 2 and 5 . This variant was not found in cancerous cells in our screening. The novel CEACAM5 variants in this study were probably cancer-specific transcripts that were processed by unpredictable alternative splicing.
The CEACAM5 splice variant involving exons 9 and 10 described in a previous report  were previously regarded as a PCR artifact . To prevent the generation of artificial amplicons, we performed validation and quantification of CEACAM5 transcript expression. Using the validated qRT-PCR assay, we found that the mRNA expression level of variant 5D was similar to that of the full-length transcript. However, in electrophoresis performed after RT-PCR, significant differences in expression levels were observed. We sequenced PCR products that were amplified using the primer pair used in Figure 1 and found several artifacts, i.e., electrophoresis bands at approximately 1,800 bp (data not shown). Therefore, we concluded that the difference between RT-PCR and qRT-PCR results likely reflects the presence of artificial amplicons.
Compared to the amino acid sequences of full-length CEA, no unique sequences were found in the novel CEA protein isoforms. Since the trypsinized peptide sequences of these isoforms are “shared” with those of the full-length protein, it is difficult to discuss the existence of isoforms based only on a typical MS/MS-based proteomic analysis. Some research groups have suggested that the combination of MS/MS-based proteomic analysis and protein separation based on molecular weight enables detection of protein isoforms that contain no unique sequences [31, 32]. However, these approaches are not suitable for the detection of protein isoforms with inconsistency between molecular weight observed in protein separation and that calculated from the amino acid sequences. Most of secreted/membrane proteins undergo posttranslational modifications, and glycosylation especially affects the results of size-dependent protein separation. In the separation approach, such proteins are not detectable at the theoretical molecular weight calculated from the amino acid sequences. Indeed, CEA is a highly N-glycosylated protein. The practical molecular weight of full-length CEA is 180–200 kDa, even though the theoretical molecular weight is 76.8 kDa [33–35]. The novel isoforms also have a large amount of N-linked oligosaccharides, which result in difference between the practical and theoretical molecular weight. The inconsistency in the molecular weight could be resolved by deglycosylation. In the present study, we proposed at strategy that avoids the influence of N-glycosylation on protein separation through enzymatic deglycosylation.
Interestingly, mRNAs of CEA were not expressed in SNU1 cells, though the bands were slightly detected by anti-CEA antibody on SNU1 cells after PNGase F treatment (Figures 4 and 6). To gain more insight into CEA-expression on SNU1 cells, condition-optimized immunoblot detection (details in figure legends in Additional file 6: Figure S5) and protein identification by LC-MS/MS analysis were performed. Bands were detected on SNU1 samples (cell lysates and conditioned medium) regardless of the PNGase F treatment, however, CEA-related peptides were not identified from those band-detected regions (data not shown). These findings indicated that the bands detected on SNU1 by the anti-CEA antibody were non-specific bands. Accordingly, mRNA and protein of CEA were considered not to be expressed on SNU1 cells.
CEA in the blood was first observed in 1969 by Thomson et al.  and now is one of the most widely used tumor markers, however, the secreted forms and secretion mechanisms of CEA remains unclear. We found that CEA protein isoforms were secreted to the conditioned medium and that these isoforms always co-existed with the full-length CEA. Interestingly, the observed secretion pattern of CEA did not correlate with the expression pattern, as determined using a commercial anti-CEA antibody. Secretion of CEA isoform 5D is higher from colorectal cancer cell lines than from pancreatic and gastric cancer cell lines. These findings suggest that CEA protein isoforms may be secreted by the specific mechanisms from certain cancer cells.
The novel CEA isoforms contain a Ig-V like domain and some IgC-like domains; two or four IgC-like domains are deleted in those isoforms (Figure 5). IgC-like domains are required for functionality of CEACAM family proteins as homophilic and heterophilic intercellular adhesion molecules . CEA-CEA homophilic interaction between an IgV-like domain and six IgC-like domains occurs in an antiparallel reciprocal manner, which is unique in this family, and it can directly influence cancer invasion and metastasis [37–39]. Those short CEA isoforms might inhibit full-length CEA-CEA homophilic interactions or mediate the heterophilic interaction with other CEACAM family proteins, leading to the influence of CEA-related intracellular signaling events. Limitations of this study are there because of poor functional analysis of novel CEA isoforms; further studies such as in vivo experiments and direct detection of those isoforms, are necessary to investigate the physiological significance of CEA isoforms.
The present study identified novel splice variants/protein isoforms of CEA, and provided direct evidence that the protein isoforms were not only co-expressed with full-length CEA but also co-secreted into culture medium by gastrointestinal cancer cell lines. CEA is a well-known serum tumor marker, however, because of its low sensitivity and specificity, the main use of serum CEA determinations is currently in the postsurgical surveillance. Discrimination between full-length CEA and its isoforms may improve the clinical utility of CEA as a tumor marker.
Availability of supporting data
The protein coding sequences of CEACAM5 splice variants 5D and 3D are deposited in DNA Data Bank of Japan (DDBJ, http://www.ddbj.nig.ac.jp/index-e.html), which are accessible through the DDBJ accession number AB852566 and AB852567, respectively.
This research was supported by the Ministry of Education, Science, Sports, and Culture; a Grant-in-Cooperation of the Regional Innovation Cluster Program 2010; and Grant-in-Aid for Young Scientists (B), 23701092, 2011 and 24701014, 2012. The authors thank Yuko Watanabe and Kaori Kanto for technical assistance of DNA sequencing and qRT-PCR, and Tomomi Ide for support of protein extraction and immunoblotting.
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