Expression of Odontogenic Ameloblast-Associated Protein (ODAM) in Dental and Other Epithelial Neoplasms

Abstract

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.

Introduction

Odontogenic ameloblast-associated protein (ODAM) is encoded by a gene originally termed EO-009 (1) that consists of ten coding exons (Figure 1) located within an ~800 kbp region of chromosome 4q13 containing the secretory calcium-binding phosphoprotein (SCPP) cluster that specifies elements involved in bone and tooth development, as well as mineralization (2). Studies of rodent tissue have revealed that ODAM is highly expressed by mature ameloblasts (3) and is present in the enamel organ and junctional epithelial cells (4). In addition to the potential role of this protein (formerly designated APin) in odontogenesis, the results of DNA microarray analyses have shown that ODAM expression is up-regulated in human cervical (5) and gastric cancer (6).

Figure 1

The deduced amino acid sequence encoded by the human ODAM gene. The signal peptide region is underlined. The amino acids are numbered as given in Reference 1 and the ten coding exons specifying the secreted form of the ODAM are shown above.

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

Patient Specimens

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).

The human #7775-1 MTE array (Clontech, Palo Alto, CA, USA) containing RNA from 76 different tissues and cells was hybridized, as previously reported (16), using a ³²P-labeled ODAM cDNA probe (13).

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).

Protein Analyses

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).

Serologic Assays

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.

Results

Molecular Evidence for ODAM Expression

We had shown previously (8) that the cDNA generated by PCR from a CEOT specimen was identical in nucleotide sequence to the coding region of the FLJ20513 gene cloned from the human Kato III signet-ring gastric carcinoma cell line (and other sources) that specified a 153-residue protein (9,10). To determine if these cells also expressed a longer ODAM transcript (4), we used a primer specific for the upstream coding exon 2 and found, by RT-PCR, cDNA specifying a 256-residue molecule that included an additional 103 amino acids located N-terminal to the FLJ20513 form and encompassing positions 24 to 126 (Figure 2).

Figure 2

Analyses of ODAM RT-PCR products derived from the Kato III human gastric carcinoma cell line. Lanes 1 and 3 contained cDNAs for exons 5–10 and 2–10, respectively, and lanes 2 and 4, full-length aldolase cDNA as an RNA template control.

Synthesis of rODAM-Related Polypeptides

To provide the requisite material for immunochemical studies, recombinant products that represented the coding sequences of either the Kato III-derived FLJ20513 or exons 2–10 of full-length ODAM were synthesized. Analysis of the two ³⁵S-labeled in vitro translated products by SDS/PAGE (Figure 3A) indicated Mrs of ~21,000 and ~38,000, respectively. These masses were higher that those predicted from their primary structure (i. e., 18,424 and 29,298) and may have resulted from protein misfolding (or other factors) that impeded gel migration. Nonetheless, mass spectrometric analyses revealed Mrs consistent with the expected values and, in the case of the long ODAM product, it was comparable to that reported for the rat homolog (4).

Figure 3

In vitro and in vivo expression of rODAM. Immunodetection of ODAM poly-peptides. (A) Fluorographs of ³⁵S-labeled translation-runoff products from exons 5–10 (lanes 1 and 2) and exons 2–10 (lanes 3 and 4) plasmid templates encoding FLAG-tagged ODAM proteins, incubated with a murine anti-FLAG IgG mAb (lanes 1 and 3) or, as a control, an irrelevant murine IgG mAb (lanes 2 and 4). (B) Western blots of exons 5–10 (lane 1) and exons 2–10 (lane 2) forms of rODAM expressed in E. coli and probed with the anti-FLAG antibody. (C) Fluorographs of ³⁵S-labeled exons 2–10 and 5–10 rODAM translation runoff-products (lanes 1 and 4, respectively) immunoprecipitated with mAbs 5A-1 (lanes 2 and 5) and 8B-4 (lanes 3 and 6).

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 [4], 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

The monoclonal anti-ODAM antibody 5A-1 immunostained CEOT and ameloblastoma cells, as well as dental epithelial cells present in unerupted human tooth follicles and ameloblasts in ten-day-old mouse teeth (Figure 4). Similar results were obtained with the polyclonal anti-ODAM antiserum (not illustrated).

Figure 4

Immunohistochemical detection of ODAM molecules in malignant and normal dental tissue. Photomicrographs of a human CEOT/ameloblastoma and an extracted supernummary tooth, as well as a developing tooth from a ten-day-old mouse. The upper panels depict sections stained with hematoxylin-eosin (H&E); the middle and lower were immunostained (immunoperoxidase reaction) with anti-ODAM mAb 5A-1 or normal mouse serum (NMS), respectively, as primary reagents (TC, calcified epithelial tumor cells; CM, calcified material containing amyloid; AM, ameloblasts; CT, connective tissue; DE, dental epithelium; OD, odontoblasts; EN, enamel). Original magnifications; CEOT, Ameloblastoma, Human tooth follicle, −200; Murine 10d tooth, ×400.

ODAM Expression by Non-Dental Normal and Malignant Tissue

Hybridization of a 70-member human tissue array, using as a probe ³²P-labeled cDNA that encodes the FLJ20513 form of ODAM, revealed a strong signal with tracheal and salivary gland-derived RNA, but only a weak reaction with stomach and fetal lung (Figure 5A). ODAM expression in the two major sites also was evidenced by the demonstration that the epithelial cells were immunostained by mAb 8B-4 (Figure 5B), as well as mAb 5A-1 and the polyclonal anti-ODAM antiserum (not illustrated). Immunohisto-chemical analyses of 35 other normal tissues revealed comparable staining of the bronchus and, to a lesser extent, ovary, bladder, ureter, endometrium, and skin. Given our detection of ODAM in the human gastric carcinoma Kato III cell line, we also analyzed human tumor tissue-specific arrays and found moderate to intense immunostaining of the nucleus and/or cytoplasm with mAb 8B-4 in 46 and 48 of the 60 breast and gastric samples, respectively, and in three of six lung specimens contained in a multi-specific array (examples are illustrated in Figure 5C). This reactivity was confined principally to adenocarcinomas and could be inhibited by the presence of the immunogen. Comparable results were obtained using mAb 5A-1, albeit with varying degrees of intensity, as well as with 8B-4 and the anti-Gli-1 antibody (Figure 6). The presence of ODAM within neoplastic breast cells also was shown by in situ hybridization using a biotin-labeled oligonucleotide probe specific for exons 2–10-encoded ODAM proteins. This reactivity could be diminished by competition with the corresponding unlabeled specific ODAM oligonucleotide, but not with a non-corresponding ODAM antisense oligonucleotide. In contrast, the probe did not appear to hybridize with normal breast tissue (Figure 7).

Figure 5

Detection of ODAM in normal and malignant tissues. (A) 70-member RNA-con-taining human tissue microarray (16) hybridized with a ³²P-labeled ODAM cDNA probe. Wells e-9, h-7, b-5, and g-11 contained tracheal, salivary gland, stomach, and fetal lung RNA, respectively. (B) Immunostained tissue sections of normal human trachea and salivary gland and (C) human breast, lung, and gastric adenocarcinomas exposed to mAb 8B-4 as the primary reagent (immunoperoxidase reaction) (CE, columnar epithelium; M, mucosa; SC, sereous cells; MC, mucosal cells; IT, intraductal tumor cells; CT, connective tissue; ATC, adenocarcinoma tumor cells; GC, gastric carcinoma cells). Original magnifications, ×200.

Figure 6

Co-localization of ODAM and Gli-1 in human epithelial malignancies. Sections of breast, lung, and gastric cancer immunostained (immunoperoxidase reaction) with anti-ODAM mAb 8B-4 and anti-Gli antibody as primary reagents (CT, connective tissue; IT, infiltrating tumor cells; LC, lung carcinoma cells; GC, gastric carcinoma cells; GL, gastric gland). Original magnifications, ×200.

Figure 7

In situ competition analyses of ODAM transcript expression in neoplastic human breast tissue. Formalin-fixed deparaffinized sections of an (A) intraductal adenocarcinoma or (B) normal breast tissue hybridized with a biotinylated antisense specific oligonucleotide probe for ODAM in the presence of 100× excess of either the non-specific (Panel 1) or specific (Panel 2) unlabeled ODAM oligonucleotides. The sections in Panel 3 were stained with pyronin Y (CT, connective tissue; IT, intraductal tumor cells; ID, interlobular duct). Original magnifications, ×200.

Serologic Evidence for ODAM Expression

To document the presence of ODAM molecules in human serum, we utilized a solid-phase capture ELISA in which wells were coated with anti-ODAM mAbs 5A-1 and polyclonal anti-ODAM that were capable of detecting this protein in amounts as low as 200 pg. Under the conditions of the assay, ODAM was not found in analyses of 24 specimens from normal individuals and 48 with epithelial forms of cancer. In contrast, antibodies to ODAM were present in patients’ sera, as evidenced by ELISA using for capture the exons 2–10 rODAM product as the solid phase antigen. Immunoassay of archival specimens from 72 females with breast cancer revealed that 25 (35%) had anti-ODAM IgG antibodies in titers > 2 standard deviations above the mean values of 45 of 48 normal women (Figure 8A). The difference between the diseased and healthy groups was statistically significant, as determined by both standard and asymmetric t-test analyses (P < 0.001). The presence of anti-ODAM autoantibodies in patient sera also was demonstrated when these molecules immune-precipitated rODAM (Figure 8B). Similarly, analyses of sera from 63 individuals with lung and seven with gastric cancer revealed that ~30% had anti-ODAM IgG antibody reactivity against rODAM > 2 standard deviation above the mean values of 27 normal subjects (data not shown).

Figure 8

Detection of serum anti-ODAM IgG autoantibodies. (A) Serum samples from 72 patients with breast cancer (BrCa) and 48 healthy females were tested for antibody reactivity to rODAM by ELISA. The mean O.D.s for the patient and control groups, 0.662 and 0.459, differed significantly (P < 0.001). The cutoff value for positives (dotted line equals O.D. 0.750) was derived from the mean of the healthy subjects plus two standard deviations. (B) Radiolabeled rODAM was subjected to immunoprecipitation with anti-ODAM mAb 8B-4, as well as with normal human sera (NHS) and sera from three of the above patients with the highest anti-ODAM antibody reactivities.

Discussion

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 (29–31); 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 (45–47). 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.