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[Cancer Research 63, 7769-7776, November 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

Aberrant Expression and Localization of Decorin in Human Oral Dysplasia and Squamous Cell Carcinoma

Abhijit G. Banerjee1, Indraneel Bhattacharyya2, William M. Lydiatt3 and Jamboor K. Vishwanatha1

1 Departments of Biochemistry and Molecular Biology,
2 Oral Biology,
3 Head and Neck Surgery, University of Nebraska Medical Center, Omaha, Nebraska


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The small leucine-rich proteoglycan decorin has been associated with negative regulation of cell growth. It has a prominent role in transforming growth factor (TGF)-ß and epidermal growth factor receptor activation pathways that contributes to its role in cellular proliferation, angiogenesis, and immunomodulation. Our studies are directed toward analysis of decorin gene expression, identified through DNA microarray studies, in oral premalignant and malignant tissues as well as representative cell lines of an oral cancer progression model.

We have used long oligonucleotide microarray analysis, immunohistochemistry, confocal microscopy, reverse transcription-PCR, sequencing, and Western immunoblot techniques to characterize decorin expression in oral premalignant archival tissues and an oral cancer progression cellular model. We have further analyzed the deduced amino acid sequence derived from full-length cDNA that do not show any deletion or mutations of the decorin expressed in oral premalignant and malignant cell lines. In our studies, we show aberrant expression of decorin in dysplastic oral epithelial cells. Both promoters P1 and P2 drive the aberrant expression resulting in exon 1a as well as exon 1b carrying transcripts. Intracellular accumulation and nuclear localization of aberrantly expressed decorin were observed in dysplastic oral tissues and in the respective cell lines.

Decorin expressed in oral cancer may have lost its ability to inhibit TGF-ß signaling and activate epidermal growth factor receptor signaling pathways because of such aberrant nuclear localization, resulting in a major dysfunction of otherwise a natural extracellular antagonist of TGF-ß and a putative tumor suppressor protein. The aberrant nuclear localization of a leucine-rich repeat protein might result in additional protein-protein interactions and resulting changes in gene expression. Further studies to characterize such interacting proteins and localization-dependent effects of aberrant decorin expressed in oral cancer progression are warranted.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Precancerous oral lesions, primarily leukoplakias and erythroplakias, present a diagnostic and therapeutic challenge to clinicians. Silverman et al. (1) followed patients with leukoplakia, a premalignant lesion, for 8 years and noted malignant transformation occurred in 17.5% of the cases. On the basis of American Cancer Society data, an estimated 29,000 new cases of oral cancer will be diagnosed in the United States in 2002 and approximately 7,400 people will die of the disease (2) . Oral cancer accounts for about 3% of cancers in men and 2% in women. The 5-year and 10-year relative survival rates are 54 and 39%, respectively. Cancers of the oral cavity occur more frequently in the African-American population than in Caucasians. However, demographics are changing with increased use of tobacco (including smokeless tobacco) and alcohol among women and young adults. Although overall incidence of oral cancer has decreased, the gender gap between men and women has narrowed (3) . The critical needs in oral cancer treatment are development of useful approaches to prevention of dysplastic lesions, increased accuracy of diagnosis, and discovery of definitive biological markers for progression of these lesions to carcinomas. The goal of our studies was to identify genetic alterations in premalignant lesions before they become clinically evident as a malignant phenotype.

The extracellular matrix plays an integral role in the biological processes of development, tissue repair, and metastasis by regulating cell proliferation, differentiation, adhesion, and migration. Experimental evidence shows that tobacco exposure alters the interaction of cell and extracellular matrix that is critical for maintenance, proliferation, differentiation, angiogenesis, and apoptosis of cells (4, 5, 6) . Solid tumor growth involves some stimulatory and inhibitory factors as well as stromal components that regulate functions such as cellular adhesion, migration, and gene expression. Tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow (7) . A distinct pattern of mRNA expression characterizes the generation of vascular stroma that plays a role not only in growth of the primary tumor but also in invasion and metastasis (7) .

Decorin is a SLRP.4 The leucine-rich repeat domain of the SLRP family is unique within the superfamily in that it is flanked by cysteine clusters, and the 24-amino acid consensus for SLRP members is X-X-I/V/L-X-X-X-X-F/P/L-X-X-L/P-X-X-L-X-X-L/I-X-L-X-X-N-X-I/L, where X is any amino acid (8) . SLRPs members such as decorin, biglycan, fibromodulin, and lumican, are not only extracellular matrix organizers but also binding partners of TGF-ß (9) . Decorin is involved in growth control through down-regulation of EGFR (10) . Decorin inhibits apoptosis in endothelial cells cultured in a collagen lattice and that the expression of decorin is a sufficient signal in EA.hy926 cells for a finely tuned induction of selected matrix metalloproteinases involved in angiogenesis (11) .

The decorin gene encodes a proteoglycan with putative structural and regulatory functions, the expression of which is markedly increased in human mesangial cells exposed to high concentrations (15–30 mM) of glucose (12) . Decorin expression was shown to be regulated by two promoters (distal P1 and proximal P2), resulting in two alternative first exons, exon 1a and 1b. Transcripts driven by both promoters were present in human mesangial cells maintained in 4 mM D-glucose-containing medium, whereas only P1 promoter was active in high glucose (30 mM) conditions and promoter P2 was shut off. Deletion analysis of promoter P1 showed that basal transcriptional activity lies within the proximal 378 bp, and the major high glucose and TGF-ß response element is located in the -683 to -583 bp region, whereas a TGF-ß inhibitory element is present in promoter P2. In addition, a putative cAMP response element-like sequence (TGACGTTT) lies within this region.

The largest known decorin message is 1751 bp consisting of eight distinct exons with very similar exon/intron boundaries. Several transcript variants are expressed in different tissues, such as A1, A2, B, C, D, and E (GenBank accession numbers NM_001920 and NM_133503 to 133507). Transcripts A1 and A2 encode the same protein isoform but have alternate 5'-UTRs arising from differential promoter activity and alternate exon splicing (13) . The variant B lacks exons 3 and 4, resulting in an internal deletion of 109 amino acids. The variant C lacks exons 3 to 5, causing a frameshift resulting in a protein that is 147 amino acids shorter as compared with isoform A. The variant D lacks exons 4 to 7, resulting in an internal deletion of 187 amino acids. The variant E also lacks exons 3 to 7, causing a frame shift and encoding an isoform 284 amino acids shorter than variant A.

The naturally occurring inhibitors of TGF-ß-dependent fibrotic response includes decorin (14) . Although the mechanism by which TGF-ß stimulates angiogenic gene expression via the Smad signal transduction pathway is becoming clear, the precise mechanism by which decorin may impinge upon TGF-ß activity remains to be established. Decorin modulates growth factor activities and growth factor distribution attributable to its TGF-ß binding properties and the interaction of core protein with other growth factors (15) . Both immobilized and soluble decorin bind to the EGFR ectodomain or to purified EGFR (16) . The binding is mediated by the core protein and has relatively low affinity (Kd, ~87 nM). Therefore, decorin is considered as a novel biological ligand for the EGFR, an interaction that regulates cell growth during remodeling and cancer progression. Binding of decorin induces dimerization of the EGFR and rapid, sustained phosphorylation of mitogen-activated protein kinase in squamous carcinoma cells. In a cell-free system, decorin induces autophosphorylation of purified EGFR by activating the receptor tyrosine kinase and can also act as a substrate for the EGFR kinase itself (17) .

In this study, we have analyzed the expression pattern of decorin gene identified through DNA microarray studies in oral premalignant and malignant tissues as well as representative cell lines of an oral cancer progression model. Our data show that decorin is aberrantly expressed in dysplastic oral epithelial cells. Transcripts from both P1 and P2 promoters are expressed and result in a decorin protein that seems to be aberrantly localized in the cell nucleus, whereas native decorin is reported be an extracellular component. Aberrantly expressed decorin may drive the progression of dysplastic cells to carcinoma because of its hitherto unknown molecular features and is likely to result in loss of negative feed back control mechanisms of TGF-ß signaling pathway.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines and Tissues.
The tissue samples that were used for our studies were obtained as biopsy specimens from oral surgical units or from patients undergoing surgical resection of tumors after obtaining informed consent according to the institutional review board guidelines. The unmatched patient samples were composed of paraffin blocks of normal tissue (n = 3), dysplasia specimens (n = 15), or tumor specimens (n = 3). Total RNA for DNA Microarray and subsequent RT-PCR analysis was obtained from frozen biopsies that included matched dysplasia (n = 1) or tumor (n = 2) specimens along with their normal adjacent tissues. The oral cell lines were obtained as gifts from Dr. N. H. Park (UCLA School of Dentistry, Los Angeles, CA; HOK16B), Dr. J. G. Rheinwald (Harvard Institutes of Medicine, Boston, MA; SCC4, SCC15, and SCC66), and Dr. S. Hsu (Medical College of Georgia, Atlanta, GA; DOK, SCC25, and OSC-2 as oral cancer progression model) and were maintained in culture as described earlier (18, 19, 20) .

RNA Isolation.
RNA was isolated using the Trizol solution (Life Technologies, Inc., Rockville, MD) from monolayer cells and frozen biopsies/surgical resection specimens as per the manufacturer’s recommendations. Further purification of total RNA was carried out with RNeasy columns (Qiagen Inc., Valencia, CA) for DNA microarray analysis or RT-PCR.

DNA Microarray Analysis.
A small-scale, long oligonucleotide array analysis was carried out with Atlas plastic array kit (BD Biosciences-Clontech, Palo Alto, CA). One µg of total RNA from normal or tumor tissue frozen biopsies of the same patient was used for cDNA probe synthesis and labeled with [{alpha}-33P]dATP (Amersham) using anchored oligo-dT primers and Powerscript reverse transcriptase. cDNA probes were purified using Nucleospin extraction columns. cDNA synthesis control reactions were carried out with phage lambda DNA and primer mix to check probe synthesis and hybridization efficiency when added to the hybridization reaction. This cDNA control probe also helps to align the spots on the plastic membrane autoradiographs for grid application and data extraction using storage phosphorimaging analysis on Storm 840 (Molecular Dynamics). The array hybridization reactions were carried out in PlasticHyb solution as per manufacturer’s protocol at 60°C for overnight with continuous rotation of hybridization bottles. The array was washed in recommended solutions, air dried, and exposed to a phosphorimaging screen in a cassette for 7 days. The phosphorimager screen was scanned at a resolution of 50 µm. The image of the hybridized arrays was also obtained on X-ray film (Biomax LS; Eastman Kodak).

Immunohistochemistry.
Paraffin-embedded tissue sections were deparaffinized with EZ-DeWax solution (Biogenex Inc., San Ramon, CA) for 10 min, followed by a 2-min rinse with deionized water. Tissue sections were washed with PBS and treated with 0.3% H2O2 in absolute methanol for 30 min to block endogenous peroxidase activity. The slides were washed three times with PBS and immersed in 2.5% normal horse serum (Vector Laboratories, Burlingame, CA) for 30 min to inhibit nonspecific binding. To reduce the amount of free avidin and biotin in the tissue, the slides were incubated in avidin-blocking and biotin-blocking solutions (Vector Laboratories) for 15 min each. The tissue sections were then incubated in primary antibody overnight at 4°C. An affinity-purified goat IgG antibody raised against recombinant human decorin was used at 1:50 dilution (AF-143; R&D Systems, Minneapolis, MN) or a polyclonal rabbit antiserum raised against decorin-specific peptide at 1:5000 dilution [LF136 (Ref. 21 )] in two separate experiments. Slides were washed with PBS and incubated for 30 min with biotinylated pan-specific secondary antibody (H+L) made in horse (Vector Laboratories). After washing in PBS, the sections were treated with streptavidin-peroxidase complex solution (Vector Laboratories) for 45 min at room temperature. Visualization of signal was achieved with incubation in a substrate solution composed of 250 µl of Tris (pH 7.5), 2.5 mg of diaminobenzidine tetrachloride and 1.7 µl (0.3% v/v) of H2O2 in 5 ml of ddH2O for up to 2 min. Sections were counterstained with hematoxylin and mounted using Vectamount permanent mounting medium (Vector Laboratories). All procedures were carried out under humidified conditions at room temperature unless otherwise specified.

RT-PCR Analysis.
Initially, 2 µg of total RNA were subjected to reverse transcription using anchored oligo-dT primers (Sigma-Aldrich Biochemicals, St. Louis, MO). The primers were annealed at room temperature for 10 min after heat denaturation of RNA and oligo-dT mix at 70°C for 15 min. Reverse transcription reaction was carried out at 50°C for 1 h; denaturation was performed at 70°C for 15 min, followed by snap cooling on ice. Subsequently, PCR amplification was conducted using HotstartTaq polymerase kit (Qiagen Inc.) with two primer sets in a multiplex reaction using specifically designed decorin (DCN) and cyclooxygenase-1 (COX-1) specific primers as internal control for normalization as per optimized protocol described below. The sequences for Decorin and COX-1 primers are: DCN-F-2, 5'-CGAGTGGTCCAGTGTTCTGA-3'; DCN-R-2, 5'-AAAGCCCCATTTTCAATTCC-3'; COX-1-F, 5'-GTTTGGCATGGTGAGTGTTG-3'; and COX-1-R, 5'-AGAAGAACACCCCACCTCCT-3'.

Briefly, the multiplex PCR reaction conditions for amplification of decorin message are as follows. Initial denaturation at 95°C for 15 min for activation of the polymerase enzyme as recommended by the manufacturer was followed by 1 cycle of annealing at 57°C for 1 min and extension at 72°C for 1 min. Subsequent 35 amplification cycles were carried out at 95°C for 1 min, 55°C for 30 s, and 72°C for 30 s with a final extension at 72°C for 3 min. PCR products were analyzed on a 1.5% agarose gel in 1x TBE (Tris-borate EDTA) at 100 V. An internal control for integrity of mRNA used for the study was checked using IL-10 primers from Maxim Biotech Inc. (South San Francisco, CA) as per the manufacturer’s guidelines.

RT-PCR for full-length decorin transcripts from oral cell lines were similarly amplified using forward primers specific to exon 1a or 1b in the 5'-UTR region of the gene preceding the start codon and reverse primer specific to 3'-UTR following the stop codon. The sequences of the primers are: DCNex1a-F1, 5'-GTGGCAAATTCCCGGATTAA-3'; DCNex1b-F2, 5'-CCAGGGGACACAGAAGAGAA-3'; and DCN1256-R1, 5'-GTCATGTGGGTAAAACATCCA-3'. The amplification cycles were initial denaturation at 95°C for 14 min, followed by 35 cycles of 94°C for 1 min, 58°C for 1 min, 70°C for 75 s, and final extension at 72°C for 5 min using HotstartTaq Polymerase (Qiagen Inc.).

Sequence Analysis.
The full-length PCR products of decorin were purified by electrophoresis on 1% agarose gel in 1x TBE. PCR products were purified from the appropriate bands excised from agarose gels followed by Qiaquick spin column purification (Qiagen Inc.). The purified PCR products were then sequenced on an Applied Biosystems sequencing apparatus using automated fluorescent thermal cycle sequencing at our molecular biology core facility.

Western Blot Analysis.
Cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 2.5 mM phenylmethylsulfonyl fluoride, 5 mM iodoacetamide, 5 mM EDTA, 150 mM NaCl, 0.5% NP40, and 0.5% Mega-9 (N-onanoyl-N-methylglucamide), 0.5 µg/ml of leupeptin, and 1 µg/ml of pepstatin for total protein lysates preparation. Alternately for cytoplasmic and nuclear fraction preparation NE-PER kit reagents (Pierce) were used as per the manufacturer’s recommendation. Protein concentration was determined by bicinchoninic acid protein assay (Pierce) and subjected to 8% SDS-PAGE analysis, followed by transfer to polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). The membrane was immunoprobed with 1:250 dilution of affinity-purified goat anti-human decorin antibody (AF-143; R&D Systems) or 1:4000 dilution of polyclonal antibody against anti-human decorin peptide (LF136). Western blots were developed with appropriate secondary antibodies conjugated to horseradish peroxidase and ECL+ chemiluminescence system (Amersham Pharmacia) and exposed to autoradiographic film for the indicated time points. Films were developed using Konica SRX-101A X-ray film developer.

Confocal Immunofluorescent Microscopy.
The oral cell lines of progression model, HOK16B, DOK, SCC25, and OSC-2, were cultured on glass coverslips until 60–70% confluence. Cells were fixed in chilled 2% paraformaldehyde solution in PBS at 4°C for 15 min and then permeabilized with 0.1% Tween 20 containing PBS for 10 min at room temperature, followed by three washes for 15 min each in PBS. Non-specific protein binding was reduced by incubation in 2% goat serum in PBS at room temperature for at least 4 h. The cells were then incubated with 1:100 dilution of rabbit primary antibody against human decorin (LF 122) in 2% goat serum containing PBS at 4°C for overnight. The cells were washed three times for 15 min each with 0.05% Tween 20 containing PBS (PBST), followed by incubation with goat anti-rabbit secondary ALEXA594 conjugate for 2 h at room temperature. Again, the cells were washed three times for 15 min each in PBST, followed by a brief rinse in deionized water to wash off salts. The coverslips were mounted in aqueous antifade reagent (Vector Laboratories) on glass slides and sealed with nail polish. Microscopic observations were made using a laser scanning confocal microscope (Carl Zeiss, Göttingen, Germany).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DNA Microarray Expression Profiling.
Our goal was to address the need for a definitive molecular marker to classify early oral lesions for their invasive potential and biological behavior. Initially, we performed a small-scale microarray analysis using mRNA from fresh frozen oral tumor and normal adjacent tissue on a set of 96 genes using ATLAS plastic microarray. Signal from a housekeeping gene (RPS 09) was used to normalize the data. Our analysis revealed 15 genes whose expression was increased in paired diseased (n = 2) relative to adjacent normal tissue (Fig. 1A)Citation samples. Of these we chose the decorin gene for further validation using RT-PCR and immunolocalization on serial tissue samples and cell lines to address its role on oral tumor progression. In addition, MARCKS, a cytoplasmic substrate of protein kinase C, and MHC-1C4{alpha}, a known immunomodulatory gene affecting immunosurveillance, were also up-regulated significantly. Other genes found up-regulated were enzymes involved in inflammation such as phospholipase A2 that also interacts with decorin (22) and an S-100 calcium binding protein A, a known marker for metastasis (23) . We also observed the same set of genes up-regulated in another DNA microarray analysis performed using Affymetrix human U133A gene chips that confirm our results (data not shown), thus indicating the putative involvement of these genes in oral dysplasia and carcinoma.



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  		Fig. 1. Microarray and RT-PCR analysis of representative differentially expressed genes. A, the images and relative ratios obtained from the microarray experiment are expressed as fold changes relative to RPS 09, a housekeeping gene. The data for significant genes known earlier [phospholipase A2 (PLA2) and S100Ca2+BPA1] and identified in this study (DCN, MARCKS, and MHC1-C4{alpha}) in oral malignancy are shown. B, RT-PCR analysis of decorin expression in oral cell lines. Lane 1, HOK16B (P = 78); Lane 2, HOK16B (P = 84); Lane 3, DOK (P = 3); Lane 4, SCC4; Lane 5, SCC15; Lane 6, SCC66; Lane 7, OSC2; Lane 8, normal mucosa 1; Lane 9, normal mucosa 2; Lane 10, verrucous carcinoma. M, 100-bp ladder (Bio-Rad). p, passage number. C, RT-PCR analysis of oral tissue specimens. Lane 1, buccal swab left cheek brush; Lane 2, buccal swab left tongue depressor; Lane 3, buccal swab right cheek brush; Lane 4, buccal swab right tongue depressor; Lane 5, normal adjacent tissue; Lane 6, dysplastic biopsy; Lane 7, oral tumor; Lane 8, HOK16B cell line; M, 100-bp DNA ladder (Life Technologies, Inc.). The internal control of IL-10 is also included to show integrity of the mRNA.

 
Decorin Transcripts in Early Oral Premalignant, Malignant Tissues and Oral Cell Lines.
Expression of decorin message was studied in oral epithelial cell lines. These cell lines represent various stages of human oral cancer progression and includes a human papilloma virus-transformed oral keratinocyte cell line (HOK16B), a dysplastic oral epithelial cell line (DOK), three squamous carcinoma cell lines (SCC4, SCC15, and SCC66) and a metastatic oral cancer cell line (OSC-2). All of the cell lines showed expression of the decorin message at various levels in our RT-PCR analysis using internal primers from exon 2 and exon 5 (Fig. 1B)Citation . The normal mucosa did not show decorin expression, indicating that decorin transcription initiates either in transformed oral cell lines (HOK16B) or oral dysplastic cells.

Early premalignant oral tissue biopsies were analyzed for decorin message by multiplex RT-PCR using decorin and COX-1 primers. It was observed that decorin transcripts are up-regulated in oral dysplasia (Fig. 1CCitation , Lane 6) as compared with very low or no expression seen in normal tissue (Fig. 1CCitation , Lanes 1–5). Decorin is expressed at relatively high levels in oral tumor (Fig. 1CCitation , Lane 7) as observed from our data, but the amplification of decorin-specific transcripts in the HOK16B cell line shows up at lower levels relative to COX-1 (Fig. 1CCitation , Lane 8), implying that decorin expression may be correlated to events after viral transformation. The buccal brush biopsies from unaffected individuals (Fig. 1CCitation , Lanes 1–4) and normal tissue from dysplasia (Fig. 1Citation C, Lane 5) were included as controls to ascertain detection sensitivity of the RT-PCR assay. IL-10-specific messages were analyzed as RT-PCR control to ascertain integrity of mRNA and cDNA from the samples. We have shown through our earlier studies that cancer-predisposing gene changes associated with oral premalignant tissues do occur in the same series of samples (24) .

Decorin Expression in Oral Dysplasia Specimens.
The paraffin-embedded oral tissue archival specimens (n = 21) were cut into 3-µm sections and processed for immunohistochemical analysis with anti-human decorin antibody as described in "Materials and Methods." As expected, the stromal tissue stained positively for decorin expression. We observed that the dysplastic cells in the basal epithelial layer stained strongly in all (15 of 15) of the dysplasia specimens (Fig. 2A)Citation . This aberrant decorin expression in epithelial cells appeared mostly in the cytoplasmic compartment. Significantly, we find localization of decorin in the nuclei of several dysplastic cells. None of the epithelial cells in normal tissue expressed decorin (Fig. 2ACitation , iv).



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  		Fig. 2. Immunohistochemical staining of oral dysplasia specimens. A, anti-human decorin staining of four representative oral biopsy archival specimens representing various grades of dysplasia (i–iii) as compared with control fibroma specimen (iv) shows abnormal cytoplasmic staining in dysplastic (arrows, panels i–iii) as against negative normal basal epithelial cells (arrows, panel iv) and positive extracellular matrix component of stromal fibroblasts as internal controls (shown encircled). B, a representative magnified image (x200) of anti-human decorin immunostained oral dysplastic epithelial cells shows the aberrant perinuclear cytoplasmic (arrows with dots) and nuclear localization (arrows) of a normally secreted extracellular matrix proteoglycan. Some atypical mitotic cells were also observed.

 
A closer look at the dysplastic epithelium revealed perinuclear cytoplasmic granular staining for decorin (Fig. 2B)Citation . Mitotic cells appearing in this area confirm cellular proliferation in the dysplastic epithelium as a cytomorphological marker of the proliferative disease within a supposedly differentiated tissue. Some of these mitotic cells also had a similar decorin expression pattern. The cellular morphology suggests possible changes from a normal epithelial cell phenotype to a spindle-shaped or more fibroblast-like phenotype. This morphological change may enable these cells to acquire cell motility and migration during transformation into an invasive phenotype. Nearly 80–90% of cells having this typical morphology stained positively with moderate to strong intensity correlated with increasing grades of dysplasia from mild, moderate, or severe, respectively. There was diminished staining intensity observed for decorin expression in malignant cells, barring few regions of intense organization of invasive squamous epithelial cells.

Decorin Protein Expression in Oral Cancer Progression Model.
Decorin protein levels were analyzed by immunoblot analysis of total cell lysates prepared from oral cell lines as described in "Materials and Methods." Proteins were separated on 8% SDS-PAGE gels, followed by immunoblotting with affinity-purified goat antiserum against recombinant human decorin or specific peptide (LF136). The decorin core protein is expected to migrate at ~51 kDa or higher. However, the Western blot profile in our studies shows protein bands migrating faster than the core protein (<49.5 kDa) in SCC25 and OSC-2 cell lines (Fig. 3A)Citation . We have consistently observed a band <49.5 kDa as the major band immunoreactive with anti-decorin-specific antibodies. The HOK16B, SCC4, SCC25, and SCC66 cell lines show multiple protein bands, including a protein band suggested for decorin variant A by Krusius et al. (25) . A similar Western blot with an antibody (LF136) raised against a specific peptide of decorin was used to detect expression of decorin in oral cell lines after chondroitin ABC lyase treatment as described in "Materials and Methods." The enzymatic treatment increased the detection sensitivity as expected but without any effect on migration of bands except that the protein bands from the OSC-2 cell line seemed less intense (Fig. 3B)Citation . To characterize the decorin molecule being expressed and sequence changes if any in the decorin transcripts, we amplified the decorin-specific message from various cell lines in the progression model as well as frozen tissue biopsies.



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  		Fig. 3. Decorin protein analyses from oral cell lines. The total protein lysates from various oral cell lines were immunoprobed with goat anti-human decorin antibody before (A) or after (B) chondroitin ABC lyase treatment. The squamous cell carcinoma (SCC25) and malignant oral cell line (OSC-2) show similar decorin variant band patterns.

 
Altered Localization of Decorin in Oral Progression Model.
Since our immunohistochemical analysis of decorin in oral biopsies showed both cytoplasmic and nuclear localization, we investigated localization of decorin in cell lines of an oral progression model (18, 19, 20) consisting of HOK16B, DOK, SCC25, and OSC-2 cells to ascertain similarity in vitro. Immunofluorescent confocal microscopy was performed on cells grown on glass coverslips as described in "Materials and Methods." The panel of progressive z-sections of indicated cell lines shows intracellular localization of immunofluorescence-tagged decorin molecules in this cell model of oral cancer progression (Fig. 4)Citation . We see mostly cytoplasmic localization of decorin (26) in HOK16B cells as compared to the perinuclear or nuclear localization of decorin in DOK and SCC25 cells. The metastatic OSC-2 cells show both cytoplasmic and perinuclear localization. This is the first report of an aberrant nuclear localization of decorin in oral dysplasia and squamous cell carcinoma.



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  		Fig. 4. Immunofluorescent confocal localization of decorin in an oral cancer progression model. Indirect immunofluorescent staining with rabbit anti-human decorin primary antibody followed by Alexa595-conjugated anti-rabbit secondary antibody shows altered localization of aberrant decorin expressed in DOK cells. The progressive z-sections observed by confocal microscopy at x63 of each cell line are represented as 1–4.

 
The representative magnified image from confocal microscopy (Fig. 5A)Citation and Western blot analysis of nuclear and cytoplasmic extracts of oral cell lines (Fig. 5B)Citation further confirm the extent of localization and identity of the molecular forms involved in the cytoplasm to nuclear shuttling. The data confirm the presence of major amounts of the decorin variant in nuclear fraction in the DOK, SCC25, and OSC-2 cells as compared with the cytoplasmic fraction, although high molecular weight (~60 kDa) cross-reactive complexes are present in both cytoplasmic as well as nuclear fractions of DOK cells. The amount of decorin in HOK16B cells seems to be a much lesser amount and mostly cytoplasmic. These data also supplement our confocal localization results discussed in the previous section in the oral cancer progression model. The expression level of decorin is markedly up-regulated in the DOK cell line. There was no alteration in migration seen for high molecular weight complexes in nuclear fraction, even after chondroitin lyase treatment. Although we have not tested, it seems that cytoplasmic to nuclear shuttling of decorin may be cell cycle dependent because we see the nuclear localization mostly correlating with the mitotic stage (rounded) of cells.



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  		Fig. 5. Cytoplasmic and nuclear localization of decorin in oral cell lines. The representative magnified images of cell lines show localization of decorin variant in nuclear compartment of DOK and SCC25 cells (A). Equal amounts of protein loaded (12.5 µg/lane) after chondroitin ABC lyase treatment show higher molecular weight complexes (~60 kDa) in the nuclear fraction (B) of all of the stages of the oral progression model.

 
RT-PCR Analysis of Full-Length Transcripts of Decorin Gene from Oral Cell Lines and Specimens.
Because the internal primer-based decorin gene expression analysis (Fig. 1, B and C)Citation did not reveal its molecular nature other than its presence in almost all stage-specific cell lines and biopsies, we investigated expression of the full-length decorin transcript to observe any exon deletions or mutations in oral preneoplastic, dysplastic, and malignant tissue-derived cell lines. RT-PCR for full-length decorin transcripts were performed using external primers corresponding to 5'-UTR specific to exon 1a or exon 1b and 3'-UTR as described in "Materials and Methods." We observed amplification of expected size PCR products of >1200 bp (exon 1a) and 1300 bp (exon 1b) upon agarose gel electrophoretic analysis (Fig. 6)Citation . These data do not suggest any major exon deletions in the transcripts per se and is unable to explain altered protein bands seen from the Western blot analysis. Some alternate PCR products were also observed, even at high stringency amplification conditions. For a check, when BLASTed against a nonredundant gene database, primer sequences used in these studies did not suggest any alternative homology within or outside the decorin gene. These further confirmed the fidelity of amplification using our primers.



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  		Fig. 6. Decorin message amplification from oral epithelial cell lines and tissue. The full-length decorin message was amplified with 5'- and 3'-UTR-specific primers to exon 1a, resulting in a major PCR product of ~1250 bp and few transcript variants (A). The samples are Generuler DNA ladder (MBI; Lane M), HOK16B (Lane 1), DOK (Lane 2), SCC25 (Lane 3), OSC-2 (Lane 4), normal adjacent tissue (Lane 5), dysplastic oral tissue (Lane 6), normal buccal biopsy-B3 (Lane 7). Similar amplification of full-length message with exon 1b-specific primers resulted in a ~1360-bp product and some transcript variants (B).

 
Sequence Analysis Confirms Deduced Sequence of the Decorin Gene.
The full-length PCR products amplified from the oral progression models either from exon 1a or exon 1b (Fig. 6)Citation were subjected to nucleotide sequence analysis for deciphering the sequence alterations to identify and define the possible functional role of decorin expressed in oral epithelial cells. The nucleotide sequences derived from DOK, SCC25, and OSC-2 cell lines were found to be homologous to transcript variants A1 and A2. The encoded human decorin protein suggests similarity in amino acid sequence to that of a core prepro immature form of decorin. However, decorin variant A has been suggested to have a negative regulatory role in cell proliferation and angiogenesis (27 , 28) . Whether the aberrantly localized (nuclear form) decorin molecule in oral epithelial cell is unable to function as a natural inhibitor of angiogenesis and tumor suppressor mechanistically or has a more proactive role in tumor promotion is not yet clear. It is expected that being a leucine-rich repeat domain containing protein, it may interact with putative nuclear factors yet to be identified. Additional experiments are in progress to identify such molecules as well as to characterize the nuclear form of decorin.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
DNA microarray analysis of normal and oral tumor tissues led us to investigate the role of the tumor microenvironment in oral premalignancy to search for early genetic changes. Although we chose to follow the proteoglycan decorin for our further studies as a known molecule involved in extracellular matrix organization, the other genes identified also gave clues to overall status related to disease progression. MARCKS is known to regulate actin structure and thus is involved in the plasticity of the cells (29) . MARCKS has been also associated with long-term depression seen in cancer patients and causes elevation of growth-associated protein (30) . MHC-1C4{alpha}, a human leukocyte antigen C allele, is known to affect innate immunity through involvement in inhibitory natural killer cell synapses (31 , 32) , whereas the decorin gene has also been linked with immunogenicity (33) . These additional genes (MARCKS and MHC-1C4{alpha}) identified through DNA microarray analysis in our studies need further validation using appropriate models. Overall, we do find expression profile changes that suggest genetic alterations not only favoring transformed epithelial cell per se but also with the capabilities to modulate immediate microenvironment that may aid in tumor progression. The present study identifies, for the first time, an aberrant decorin expression in premalignant and malignant tissues, suggesting its appearance and overexpression in oral dysplasia as a possible biological marker of imminent progression. The mechanism by which decorin expressed in DOK cells localizes to the nucleus is unclear, and whether decorin shuttles between the nuclear and cytoplasmic compartment is also not known. It is possible that additional interactions with other cytosolic and nuclear factors may result from the aberrant localization and result in some of its tumor-promoting role in oral cancer. Elucidation of such interactions of the aberrant decorin expressed in the oral cancer progression model and its biological implications needs further investigation.


   ACKNOWLEDGMENTS
 
We acknowledge the generous gift of decorin antibodies (LF136 and LF122) and cDNA from Dr. Larry Fischer of the Department of Intramural Research, National Institute of Dental and Craniofacial Research, NIH (Bethesda, MD). We thankfully acknowledge the assistance of UNMC Eppley Cancer Center tumor bank and molecular biology core facilities (National Cancer Institute Cancer Center Grant T30CA36727) in our studies. We also thank the UNMC confocal microscopy core facility (supported by Nebraska Research Initiative grants) for assistance.


   FOOTNOTES
 
Grant support: Philip Morris USA Incorporated.

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.

Requests for reprints: Jamboor K. Vishwanatha, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, 984525 Nebraska Medical Center, Omaha, NE 68198-4525. Phone: (402) 559-6663; Fax: (402) 559-6650; E-mail: jvishwan{at}unmc.edu

4 The abbreviations used are: SLRP, small, leucine-rich extracellular chondroitin/dermatan sulfate proteoglycan; TGF, transforming growth factor; EGFR, epidermal growth factor receptor; UTR, untranslated region; RT-PCR, reverse transcription-PCR; COX, cyclooxygenase; IL, interleukin; SCC, squamous cell carcinoma. Back

Received 6/ 3/03. Revised 7/29/03. Accepted 9/ 2/03.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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