Expression of Odontogenic Ameloblast-Associated Protein (ODAM) in Dental and Other Epithelial Neoplasms
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We previously have communicated our discovery that the amyloid associated with calcifying epithelial odontogenic tumors is composed of N-terminal fragments of the structurally novel odontogenic ameloblast-associated protein designated ODAM. Subsequently, it was shown by other investigators that ODAM is expressed in rodent enamel organ and is likely involved in dental development. We now report that this molecule also is found in certain human tissues, principally the salivary gland and trachea, as evidenced by RNA array analysis and immunohistochemistry-utilizing antibodies prepared against synthetic ODAM-related peptides and recombinant protein. Notably, these reagents immunostained normal and malignant ameloblasts and other types of human neoplastic cells, including those of gastric, lung, and breast origin where the presence in the latter was confirmed by in situ hybridization using gene-specific molecular probes. Moreover, significant titers of anti-ODAM IgG antibodies were detected in the sera of patients with these malignancies. Our studies have provided the first evidence in humans for the cellular expression of ODAM in normal and diseased states. Based on our findings, we posit that ODAM is a developmental antigen that has an essential role in tooth maturation and in the pathogenesis of certain odontogenic and other epithelial neoplasms; further, we suggest that ODAM may serve as a novel prognostic biomarker, as well as a potential diagnostic and therapeutic target for patients with breast and other epithelial forms of cancer.
Although ODAM-related gene transcripts have been detected in a variety of mammalian tissues, including those of human, chimpanzee, mouse, rat, and canine origin (7), our report that the amyloid associated with calcifying epithelial odontogenic tumors (CEOT) is formed from an ODAM molecule provided the first example of this protein’s expression (8). Through chemical analyses of amyloid fibrils isolated from three specimens, we found that these components were composed of N-terminal fragments of a 153-residue hypothetical protein specified by the FLJ20513 gene (a short form of ODAM cDNA cloned from the Kato III human signet-ring gastric carcinoma cell line [9,10]), where the last six of ten protein-encoding exons of ODAM are located. Subsequently, in studies of amyloid from four other CEOT cases, we identified varying amounts of a second ODAM-related protein with residues originating from the fourth exon (11). This discovery provided conclusive evidence for the transcription of a longer ODAM product that was predicted to consist of 279 amino acids, 126 of which are products of the first four coding exons (1,4).
To gain further insight into the potential role of ODAM in tissue development and carcinogenesis, we have generated anti-ODAM specific polyclonal and monoclonal antibodies, using as immunogens synthetic peptides and recombinant protein. We now report that these reagents recognized ODAM molecules, not only in ameloblasts, but also in certain normal and neoplastic human epithelial tissues—a finding confirmed at the molecular level through RNA array and in situ hybridization analyses. Our studies have provided definite evidence that ODAM is expressed under physiologic and pathologic conditions and suggest that when up-regulated, this protein may serve clinically as a novel cancer biomarker and provide a therapeutic target for patients with certain epithelial malignancies.
Materials and Methods
Serum samples were obtained from healthy adults, as well as patients with breast, lung, or gastric cancer, and kept frozen at −20° C prior to analysis. A maxillary hybrid tumor consisting of CEOT and an ameloblastoma was furnished by Philip Seim (12) and a supernummary tooth follicle by John Hudson. The study was conducted in accordance with a protocol approved by the University of Tennessee Graduate School of Medicine’s Institutional Review Board.
Cells and Tissues
The human signet-ring gastric carcinoma Kato III cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were grown in RPMI supplemented with L-glutamine, penicillin/streptomycin, and 10% FBS at 37° C in a humidified incubator with a 95% air and 5% CO2 atmosphere at densities of 0.1-1 × 106 cells/mL.
Human normal and malignant tissue arrays (breast, stomach, and lung) were purchased from Zymed-Invitrogen (San Francisco, CA, USA) and a multi-tissue sausage array from BioGenex (San Ramon, CA, USA). Mouse ten-day-old fixed, embedded odontogenic tissue was kindly provided by J Timothy Wright (University of North Carolina School of Dental Medicine, Chapel Hill, NC, USA).
RNA Isolation and RT-PCR
RNA was extracted from Kato III cells with guanidine isothiocyanate and cDNAs prepared by a two-step RT-PCR procedure using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), Taq polymerase (Eppendorf, Westbury, NY, USA) and, for ODAM synthesis, both coding exon 2 sense (5′-ATGTCTGCCAGCAATAGCAAT) and exon 10 antisense (5′TGGTTCCC TTAGGCTGTCAGT) primers which yielded a product encoding amino acid residues 25–279. To obtain by PCR a cDNA specifying the shorter (i.e., 153-residue) FLJ20513 product, exons 5 sense (5′ATGCCCTATGTATTCTCC) and 10 antisense primers were utilized. Aldolase-B sense and antisense primers began, respectively, at the start (5′-ATGCC CTACCAATATCCAGCACT) and termination (5′-TTAATAGGCGTGGTTAGA GACG) of the coding sequence. The reactions were run on an MJ Research model PTC-200 (Bio-RAD, Richmond, CA, USA) PCR apparatus for up to 40 cycles of amplification, each consisting of 1 min at 94, 62, and 72° C, followed by a single 15-min incubation cycle at 72° C prior to agarose gel analysis.
For expression of recombinant protein generated from the ODAM PCR products, the constructs included forward primers encoding the first methionine residue specified by the second and fifth coding exons (5′-ATGTCTGCCAGCAATAGCAAT and 5′-ATGCCCTATGT ATTCTCC, respectively) and an exon 10 reverse primer (5′-TGGTTCCCTTAGGCTGTCAGT) containing the last seven ODAM residues. For optimal prokaryotic protein production, detection, and purification purposes, the constructs also included a ribosomal binding site, a C-terminal FLAG peptide, and a termination codon.
ODAM cDNA was prepared from PCR-generated fragments rendered blunt using the Novagen (Madison, WI, USA) Perfectly Blunt Cloning Kit end conversion reagents and were ligated to blunt end plasmid vectors with T4 DNA ligase and then cloned into E. coli (plasmid vectors pSTBlue-1 [Novagen] and pSmart [Lucigen, Middleton, WI, USA]). The recombinant products were detected by direct PCR screening or Southern analysis of colony lifts using ODAM-specific cDNA probes (13) and their nucleotide sequences confirmed by automated dideoxy sequencing at the University of Tennessee’s Molecular Biology Core Facility.
Coupled transcription-translation reactions were run in 20-µL volumes in the presence of purified rODAM plasmid constructs (25 µg/mL) and 35S-Trans Label (3000 Ci/mMole-Cys/Met, MBP, Irvine, CA, USA) using a T7 RNA polymerase reticulocyte lysate coupled transcription-translation system (Promega, Madison, WI, USA). Aliquots (1 µL) of the 35S-labeled products were used in immunoprecipitation assays and the resultant precipitates analyzed by SDS/PAGE fluorography, as detailed elsewhere (14). The rODAM constructs in BL21 (DE3) E. coli were induced in LB broth with 0.1 M isopropylthioguanine (IPTG) at 37° C for 3 h with shaking and protein expression documented by Western blotting. Recombinant molecules were isolated and purified from lysates of IPTG-induced rODAM cultures using a FLAG-affinity column (Sigma-Aldrich, St. Louis, MO, USA) and size-exclusion chromatography.
In situ RNA Hybridization and Multiple Tissue Expression (MTE) Array Analysis
Deparaffinized tumor sections, where the presence of cellular RNA was documented by pyronin Y staining, were first heated at 90° C for 20 min in 0.5× SSC (Standard Saline Citrate-0.3M magnesium chloride, 30 mM sodium citrate, pH 7) and then pre-hybridized at 42° C for 2 h in a humidified chamber with 125 µLof complete hybridization mix (Fisher Scientific, Norcross, GA, USA) containing 5× SCC, 0.5% SDS, 50% formamide, and 10% dextran sulfate supplemented with 250 µg/mL yeast tRNA (Ambion, Austin, TX, USA), 100 µg/mL sonicated, denatured herring sperm DNA (Promega, Madison, WI, USA), 0.05% Na-pyrophosphate, and 0.2 µM of a random 15-mer deoxy-oligomer. Next, a 5′-biotinylated antisense ODAM-specific oligonucleotide probe in hybridization buffer (final concentration, 0.2 µM) was added, and the incubation continued overnight at 42°C. Biotinylated and unlabeled 30-mer antisense oligonu-cleotide probes from coding exon 2 (5′-CA-GAGAAAGGTGGAATCCATGAATTAAGTG) and a 31-mer antisense oligonucleotide distinct from that derived from coding exon 5 (5′-CATGTAAACTGGATAGTATTGAAACATCTGT) were used in competition experiments to ensure hybridization specificity. The biotinylated ODAM probe reacted in the presence of a 100× molar excess of either the matched unbiotinylated or the non-related ODAM antisense oligonucleotide. After hybridization, the slides were rinsed with 2× SSC containing 0.1% pyrophosphate (SSCPP) and then incubated at 42° C for 30 min with 125 µL of supplemented hybridization mix containing no probe. After two successive washes at 42° C for 10 min with 2× SSCPP and then 0.2× SSCPP, the sections were exposed to HRP-labeled antibiotin IgG (Dako, Carpenteria, CA, USA) and developed for color signal using tyramide amplification (15).
Antibodies and Immunoassays
Anti-ODAM mAbs were generated by immunizing mice with a synthetic HPLC-purified peptide encompassing 45 amino acids encoded by ODAM exon 4 (Keck Biotechnology Resource Laboratory, Yale University, New Hampshire, CT, USA), as well as with the shorter rODAM molecule (residues 127–279). A polyclonal antiserum made to a 12-mer synthetic peptide (residues 67–78) was raised in rabbits (Syn-Pep Corp, Dublin, CA, USA); the anti Gli-1 antibody was obtained from Santa Cruz Biotech (Santa Cruz, CA, USA). Immunoprecipitates of 35S-labeled cell lysates and Western blotting were analyzed, respectively, by fluorography and horseradish peroxidase (HRP)-based chemiluminesence, as described previously (14,17).
For immunohistochemical analyses, 4-µm-thick formalin-fixed paraffin-embedded tissue sections were mounted on poly-L-lysine-coated slides, dried overnight at room temperature, and deparaffinized. Sections were first immersed in the Glyca pH 4.0 antigen-retrieval solution (BioGenex, San Ramon, CA, USA), heated in a microwave oven, and then immunostained using the ImmPRESS polymerized reporter enzyme-linked system (Vector Laboratories, Burlingame, CA, USA) (15).
SDS/PAGE was performed under reducing conditions in 10% tris-glycine gels (Invitrogen, Carlsbad, CA, USA). Mass spectrometric studies were done as described (8) using an ion-spray PE-Sciex type 150 EX instrument. Mass data were analyzed with Bio Multiview software provided by the manufacturer (Applied Biosystems, Foster City, CA, USA).
The immunoassay for measurement of serum anti-ODAM antibodies involved coating wells in a standard 96-well microtiter ELISA plate with 100 µL of the full-length rODAM (5 µg/mL in PBS). The wells were washed, blocked with BSA, filled with 100 µL of patients’ serum diluted 1:50 in PBS/Tween/BSA for 2 h at room temperature, and then washed with PBS/Tween. Next, biotinylated goat anti-human IgG (in ELISA diluent) was added to each well and, after 1 h and a wash, the wells were filled with HRPlabeled streptavidin. Following a 1 h incubation and a final wash, the ABTS/H2O2 substrate (KPL, Gaithersburg, MD, USA) was added and the color measured by absorbance photometry 30 min later in an ELISA plate reader at 405 nm using a Wallac 1420 Multilabel Counter (Perkin Elmer, Shelton, CT, USA). Sera were tested in duplicate and population statistics analyzed for significance using Graph Pad Prism 4 software.
Molecular Evidence for ODAM Expression
Synthesis of rODAM-Related Polypeptides
Immunoprecipitation and Western blot studies indicated that the products contained the C-terminal FLAG epitope (Figure 3B). Shorter, immunoreactive proteins corresponding in size to that encoded by exons 5–10 were consistently observed in both in vivo and in vitro products obtained from the larger rODAM construct (these components presumably represent partial degradation products, as noted by other investigators , and/or secondary initiation sites).
Generation of Anti-ODAM Antibodies
As part of an effort to develop antibodies that would recognize ODAM-related molecules, synthetic peptides and protein were used as immunogens. A polyclonal antiserum was obtained from a rabbit immunized with a 12-mer peptide (residues 67 to 78). In addition, two IgG mAbs were generated: one, designated 5A-1, resulted from immunizing mice with a peptide encompassing residues 52 to 90 in coding exon 4, and the other, 8B-4, was produced against the 153-amino acid FLJ20513 form of rODAM. As illustrated in Figure 3C, mAb 8B-4 immune-precipitated both exons 2–10 and 5–10 rODAM runoff translation products. As expected, the 5A-1 mAb did not react with the shorter rODAM molecule corresponding to FLJ20513. To localize the region of ODAM recognized by mAb 8B-4, we tested by Western blotting the reactivity of this reagent with proteolytically-derived rODAM fragments. These included molecules spanning residues 178–279, 217–279, and 223–279. This antibody reacted only with the 178–279 component, indicating that the epitope was located between ODAM residues 178 and 216 (data not shown).
ODAM Expression by Normal and Malignant Dental Tissue
ODAM Expression by Non-Dental Normal and Malignant Tissue
Serologic Evidence for ODAM Expression
The fact that the ODAM gene is located in the SCPP cluster on chromosome 4q13 implies that this element also is involved in tooth formation. Indeed ODAM-related transcripts, highly homologous to the human counterpart, have been identified in mouse and rat enamel organs (1,2,4). Our immunohistochemical studies with mAb 5A-1 have demonstrated the presence of ODAM within ameloblasts in ten-day-old murine teeth, unerupted human tooth follicles (a precursor to dental tumors, for example, CEOT) (18), and ameloblastomas. These findings indicate that this molecule has an essential role in normal and tumor-related odontogenesis. Notably, the follicular dendritic cell secretory protein gene, FDC-SP, located adjacent to ODAM, also has been found to be functional in late dental development within the periodontal ligament (19). Signaling pathways operative in odontogenesis include Shh, BMP, FGF, and Wnt that have been shown to be active in dental enamel knot signaling centers present within developing teeth and also in odontogenic tumors (20,21). Similarly, these pathways are considered to be essential in embryogenesis and organogen-esis. Recently, it has been revealed by in situ hybridization (3) and immunohisto-chemistry (4) that ODAM is strongly expressed in maturation-stage rat incisor ameloblasts and in the junctional epithelium attached to the enamel of erupted molars, as well as in the late stage of ameloblast-lineage cell cultures (3,4).
Additional products encoded by genes within the 4q13 SCPP cluster and implicated in dental development include amelogenin, amelotin, and ameloblastin (22,23). Many of these molecules, like ODAM, contain few or no cysteines and are characterized by a relatively large number of disorder-promoting amino acids, such as proline and glutamine (which, in the case of ODAM, comprise 33 and 40 of the 264 residues of the mature protein, respectively) that can impart multi-functional properties to such molecules (24) and may account for the presence of ODAM transcripts in odontogenic and non-odontogenic mammalian tissues. As for the latter, ODAM mRNA has been seen in rodent salivary and nasal glands (4) and, as we have shown, it (and protein) is present in human trachea and salivary gland. Other investigators have found this entity in the lacrimal gland (25) and breast ducts (26). Further, homologous ODAM cDNAs have been detected in fetal heart and lung, as well as in colon tissue (27).
Historically, the first documented evidence of ODAM expression resulted as part of the Japanese NEDO human genome sequencing project where FLJ20513 cDNA encoding the 153-residue ODAM component was synthesized using mRNA from the Kato III human signet ring gastric carcinoma cell line (9). This transcript (AK000520.1) encompassed coding exons 5–10 of the long form of ODAM, but did not contain a 5′-non-coding sequence (9). However, a murine ODAM cDNA (AK009298) (28) was found to have a different coding sequence at the end of the 5′ region, thus supporting the presence of alternative splicing or, possibly, a 5′ untranslated region (UTR). Seemingly, this short ODAM protein would be deficient in a secretory signal sequence and presumably would remain within the cell where it could have a more restrictive or differing functional role from that of the larger ODAM product. Notably, the gene encoding ODAM has been predicted to evolve from those specifying enamelin and other evolutionarily-derived dental components, for example, amelogenin and amelotin, where alternative splicing occurs (29, 30, 31); this phenomenon has been documented in over 60% of human and mouse genes (32).
Our studies have confirmed the presence of ODAM in gastric cancer where we demonstrated immunohistochemically that anti-ODAM mAb 8B-4 immunostained, with varying degrees of intensity, all but 14 specimens in a 60-member human gastric tumor array. Through serial analyses of gene expression and quantitative RT-PCR of four primary human stomach cancers, it was found that ODAM was one of nine genes over-expressed, as compared with non-gastric tissues, and posited that these elements could serve as possible tumor markers (33). Our detection of ODAM in other epithelial malignancies, for example, breast, lung, and gastric cancers, also suggests that this protein is up-regulated in these cancers. Interestingly, the FDC-SP gene, adjacent to ODAM in the SCPP cluster, is expressed in late dental development, as well as in neoplasms of the breast; further, it has been included as a gene signature screening component indicative of breast cancer invasiveness (19,34,35). In the case of colon cancer, this neoplasm results from a progressive series of steps whereby epithelial dysplasia evolves into a flat adenoma, carcinoma in situ, and finally, adenocarcinoma. Of note is that ODAM expression is increased ~6-fold in the earlier stage of disease, i.e., flat adenoma, as compared with the normal mucosa (36). Notably, epidermal growth factors such as ampiregulin, epigen, and epiregulin, which are found in a variety of human cancers, including those of breast and gastrointestinal origin, map to the identical chromosomal band (4q13.3) and are present in a cisoriented cluster on the same DNA strand as ODAM and FDC-SP (37).
ODAM may have a functional role in the pathogenesis of epithelial malignances, given that this molecule has been shown to interact with parvalbumin (an EF-hand calcium-binding protein) and additionally, modulates the expression of at least one metalloproteinase (MMP-20), molecules implicated in aggressive and metastatic cancer (38,39). Further, we posit that the ODAM gene is up-regulated by the Shh pathway which is involved in carcinogenesis, as evidenced by the concomitant expression of the zinc finger transcription factor Gli-1 (an Shh pathway member) in CEOT and other odontogenic neoplasms (7,40), as well as gastric (41), breast (42,43), and lung cancers (44). The discovery that ODAM, in addition to its role in the development of teeth and other body tissues, may be implicated in neoplastic transformation has potential diagnostic and therapeutic relevance.
Autoantibodies frequently are found in the sera of patients with malignances and the titers of these components have been correlated to survival and other clinicopathological parameters (45, 46, 47). Notably, the reactivity of these molecules may be directed against cancer-associated proteins (for example, Her-2, Nectin-4, Muc-1) that have been implicated in either tumor growth or invasiveness (48,49). In this regard, our detection of serum anti-ODAM IgG antibodies in patients with breast, lung, and gastric cancer implies that they represent an immune response that may have functional import. Further, the presence and titer of anti-ODAM antibodies could serve as a unique prognostic biomarker for patients with epithelial forms of cancer.
We thank P Seim for furnishing the CEOT/ameloblastoma specimen, J T Wright for the mouse dental tissue, J Hudson for the supernummary tooth follicle, Elliot K. Swab for technical assistance, and Keira Clark for manuscript preparation. This work was supported, in part, by USPHS Research Grant CA-10056 from the National Cancer Institute and the University of Tennessee Medical Center’s Physician’s Medical and Education Research Foundation.
- 9.Sugano S, et al. 2000. Hypothetical protein FLJ20513 (FLJ20513 mRNA), Accession AK000520, https://doi.org/www.ncbi.nlm.nih.gov/ Updated September 12, 2008.
- 28.Adachi J et al. (2000) Direct submission, Accession AK009298, https://doi.org/www.ncbi.nlm.nih.gov/ Updated February 11, 2008.