Clinical Cancer Research Joint Metastasis Research Society-AACR Conference on Metastasis Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nash, M. A.
Right arrow Articles by Freedman, R. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nash, M. A.
Right arrow Articles by Freedman, R. S.
Clinical Cancer Research Vol. 8, 1754-1760, June 2002
© 2002 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

The Expression of Decorin in Human Ovarian Tumors

Michael A. Nash, Michael T. Deavers and Ralph S. Freedman1

Departments of Gynecologic Oncology [M. A. N., R. S. F.] and Pathology [M. T. D.], The University of Texas M. D. Anderson Cancer, Houston, Texas 77030


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Purpose: The purpose of this study was to examine ovarian cancer cells for the expression of decorin, a proteoglycan component of the cell matrix that can inhibit cancer cell growth.

Experimental Design: Cultured ovarian cancer cells and surgically excised tumors were examined by immunohistochemistry and Western blot analysis for decorin expression. Reverse transcription-polymerase chain reaction analysis was used to analyze cultured cells for decorin transcripts.

Results: We detected decorin transcripts in two ovarian cancer cell lines by reverse transcription-polymerase chain reaction analysis. However, no decorin was found in conditioned culture medium from those cell lines. Cells treated with the proteasome inhibitor MG132 showed strong perinuclear staining with a decorin-specific monoclonal antibody by immunohistochemistry. Also, Western blot analysis showed the presence of a ladder of decorin-specific bands that were intensified by treatment with MG132, suggesting that de novo synthesized decorin was degraded by the ubiquitination pathway. The decorin component of tumor stroma was previously shown to contain high levels of chondroitin sulfate as opposed to dermatan sulfate side chains, and those molecules contained unusually high levels of O- and 6-sulfate linkages. We provided immunohistochemical evidence that these chondroitin sulfate side chains may have been produced by myofibroblasts.

Conclusions: Decorin protein expression was not detected in ovarian cancer cells. Decorin transcripts were produced and probably translated, but the protein was probably degraded by the ubiquitination pathway. We present evidence that stromal decorin of ovarian tumors was made by myofibroblasts. We also propose that decorin may be a tumor suppressor gene that is inactivated during epithelial cell development.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
Decorin is a small, leucine-rich proteoglycan component of the normal cell matrix. It consists of an N-glycosylated Mr 40,000 protein core to which a single glycosaminoglycan side chain made up of dermatan sulfate or chondroitin sulfate is attached. The side chain is extended from a common linker of xylose-galactose-galactose-glucuronic acid bound to the protein core at a serine residue. The dermatan sulfate chain is made of repeats of sulfonated dimers of D-galactosamine and D-galactosamine or L-iduronic acid, whereas chondroitin sulfate is composed of repeated dimers of D-galactosamine and D-glucuronic acid. Sulfate linkages in chondroitin sulfate may vary, i.e., O-chondroitin sulfate, 4-chondroitin sulfate, and 6-chondroitin sulfate isomers have all been described (1) .

Decorin is an effective inhibitor of tumor cell growth. Ectopic decorin was shown to inhibit the growth of colon carcinoma cells (2) , glioma (3) , and ovarian cancer cells (4) , as well as tumors of other histiogenic origins (5) . Growth inhibition was shown to be caused by up-regulation of the cyclin kinase inhibitor p21Waf1/Cip1 (4, 5, 6) resulting from decorin binding to and activating the epidermal growth factor receptor (7, 8, 9) .

Decorin is not expressed in tumor cells; however, decorin is strongly expressed in the stroma of colon (10) and breast tumors (11) . Given the tumor-inhibitory activity of decorin, it might appear that the higher levels of decorin in peritumoral stroma represent an effort by the host to contain the growth of the tumor. However, it was also shown in head and neck tumors that stromal decorin expression was reduced at the site of aggressive tumor growth (12) , which may mean that not all tumor types express the same form of stromal decorin. Also, decorin in the stroma of colon tumors contained high levels of chondroitin sulfate side chains (10) . This finding in patients may be significant because decorin-expressing dermatan sulfate side chains were 20-fold more effective than decorin-expressing chondroitin sulfate side chains in inhibiting the migration of bone tumor cells (13) . Also, the stromal decorin of colon tumors showed a higher percentage of O- and 6-sulfate linkages than that found in normal tissue (10) .

The peptide portion of decorin can bind to and inhibit the activity of TGF-ß2 (14) . Bound decorin blocks the activity of TGF-ß by preventing it from binding with its receptor complex (15) . However, side chains, when present, were shown to inhibit this activity to some degree (15) . The therapeutic potential gained by the inhibition of TGF-ß function by decorin was illustrated by showing that ectopic decorin could block the growth of glioma cells in rats (3) and reduce chemoresistance to syngeneic breast tumors in mice (16) . Interestingly, decorin obtained from bone, which contains only chondroitin sulfate side chains (17) , appeared to stimulate the activity of TGF-ß (18) . Thus, the side chains of stromal decorin may not only be important in regulating the migration of tumor cells (13) but may also have important roles in regulating other functions essential for tumor growth as well.

The observation that some tumors produce stromal decorin that is different from decorin found in normal tissue (10) leads us to question whether the tumor actually dictates to the stromal cells what type of decorin side chains to produce, or whether migration of a particular type of cell(s) into the developing tumor (cells preprogrammed to make decorin with high chondroitin sulfate composition and high numbers of O- and 6-sulfate linkages) is a more likely explanation. Fibrosis is a common feature in many tumors, including ovarian cancer. Myofibroblasts are common to many types of fibrosis and have been identified in fibrotic tumors (19) . Myofibroblasts infiltrate areas containing high concentrations of GM-CSF or TGF-ß (20) , two cytokines that are produced by ovarian cancer cells (21, 22, 23) . In the lung, myofibroblasts were attracted early in the development of fibrotic lesions by TGF-ß that was induced in macrophages by overexpressed GM-CSF (24) . Also, both decorin and myofibroblasts were shown to coaccumulate in fibrous crescents in crescentic glomerulonephritis (25) . These observations have led us to speculate that myofibroblasts may be the source of the unique decorin found in the stroma of certain types of tumors.

To our knowledge, there has been no previous investigation on the expression of decorin in ovarian cancer. Decorin has been reported to be absent in other epithelial cancers, such as breast and colon cancer, whereas enhanced decorin was reported in the stroma of these tumors (10 , 11) . The experiments described in this report were performed to determine whether the expression of decorin in the microenvironment of ovarian tumors was different from or similar to its expression in other epithelial tumors such as breast and colon cancer, i.e., whether decorin is expressed by ovarian cancer cells, and what type of decorin was expressed in the stroma of ovarian tumors. Also, we sought to see whether myofibroblasts were involved in the synthesis of the chondroitin sulfate decorin side chains found in tumor stroma.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Human Tissue.
Ovarian tissues used in these experiments were from a tissue bank maintained by the Ovarian Cancer Research Project, The University of Texas M. D. Anderson Cancer. All specimens were obtained using institutional review board-approved consent procedures.

Tissue Culture.
The 2774 ovarian cancer cell line was obtained originally from Dr. J. Sinkovics. The cell lines MCR-5 (human embryonic fibroblast), SKOV3 (ovarian cancer), and SW480 (colon cancer) were obtained from the American Type Culture Collection. All cell lines were grown in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% fetal bovine serum. The proteasome inhibitor MG132 was obtained from Calbiochem (La Jolla, CA) and stored as a 25 mM stock in DMSO at -25°C.

Antibodies.
Mouse IgG raised against 6-sulfate chondroitin and mouse monoclonal IgG raised against human decorin isolated from ovarian fibroma were purchased from Seikagaku Corp./Associates of Cape Cod, Inc. (Falmouth MA). Unlike some antibodies raised against decorin peptides, this antibody equally recognizes core proteins with or without side chains. Mouse monoclonal IgG raised against human smooth muscle {alpha}-actin conjugated to HRP was obtained from DAKO Corp. (Carpinteria, CA). Mouse IgG prepared against GAPDH was obtained from Chemicon International, Inc. (Temecula, CA), and HRP-conjugated goat antimouse IgGs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Extraction of RNA and Protein from Cultured Cells.
Nearly confluent cell cultures grown in 25-cm2 culture flasks were rinsed with PBS and lysed with guanidinium thiocyanate-phenol-chloroform, and the phases were separated by centrifugation (26) . The upper phase was precipitated with isopropanol, and the pelleted RNA was washed with 75% ethanol, suspended in diethylpyrocarbonate-treated water, and stored at -80°C. Protein was extracted from cells cultured in 25-cm2 culture flasks by scraping the cells into prewarmed SDS sample buffer (27) , boiling the samples for 5 min, and storing them at -25°C.

RT-PCR Detection of Decorin Transcripts in Ovarian Cancer Cell Lines.
RNA was reverse-transcribed overnight into DNA as described previously (28) . The cDNAs were amplified in a GeneAmp 9700 thermocycler (PE Applied Biosystems, Foster City, CA) in 50-µl reaction mixtures containing 0.25 unit of Taq polymerase (Roche), as recommended by the supplier. Primers specific for decorin (forward primer, 5'-AGGGCTCCTGTGGCAATT-3'; reverse primer, 5'-TCAGATGACCGCTGTTGG-3'; Ref. 29 ) were used in these experiments. These primers were designed to cross intron boundaries and thus prevent amplification of genomic DNA. Amplification was carried out for 35 cycles of 0.5 min at 94°C, 0.5 min at 55°C, and 1 min at 72°C, followed by a final incubation for 7 min at 72°C. Reaction products and DNA molecular weight marker VI (Roche Molecular Biochemicals, Indianapolis, IN) were loaded onto a 1.8% agarose gel in Tris-acetate-EDTA buffer (27) containing 0.25 µg/ml ethidium bromide. The gel was photographed under UV light.

Western Blot Analysis of Decorin Expression in Tumor Cell Lines.
Western blots were performed as described previously (4) . Briefly, aliquots of protein and prestained molecular weight markers (Life Technologies, Inc., Gathersburg, MD) were separated on a 10% polyacrylamide mini-gel (Bio-Rad Laboratories, Hercules, CA) and transferred onto Hybond enhanced chemiluminescence nitrocellulose (Amersham Life Sciences, Arlington Heights, IL) with a semidry transfer unit (Bio-Rad Laboratories). The blots were blocked with 5% nonfat dry milk and probed with either antidecorin or anti-GAPDH monoclonal IgG in blocking solution. The blots were treated with HRP-conjugated antimouse IgG in blocking solution, soaked in luminol (Kirkegaard and Perry Laboratory, Gaithersburg, MD), and exposed on Hyperfilm enhanced chemiluminescence X-ray film (Amersham).

Immunohistochemical Staining for Decorin in Ovarian Cancer Cells.
Cryopreserved tumors were sectioned as described previously (21) . The sections were fixed with room temperature acetone and blocked first with 0.3% hydrogen peroxide in methanol and then with 1% of the appropriate normal serum in PBS. The sections were incubated for 2.5 h with a primary antibody and for 1 h with a secondary (biotin-conjugated) antibody at room temperature. The sections were stained with the avidin-biotin-peroxidase complex vector staining kit (Vector Laboratories, Burlingame, CA) following the manufacturer’s directions. Color was developed with NovaRed substrate (Vector Laboratories). The sections were counterstained with Mayer’s modified hematoxylin (Polyscientific, Bayshore, NY) and Scott’s bluing reagent (STAT Lab Medical Products, Inc., Lewisville, TX).

Also, cultured ovarian cancer cells were grown on an 8-chambered Lab-Tek microscope slide (Nalge Nunc International, Naperville, IL). The cells were washed with PBS, fixed with room temperature acetone, and stored at -25°C until use. The slides were stained as described above, except that no counterstain was used.

Immunohistochemistical Staining for 6-Sulfate Chondroitin or Smooth Muscle {alpha}-Actin.
Paraffin-embedded sections of ovarian tissue were renatured in a microwave oven for 10 min and stained with the appropriate antibody as described above.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
RT-PCR Detection of Decorin-specific Transcripts in Cultured Ovarian Cancer Cells.
When we amplified cDNA made from human fibroblasts, we observed a predominant transcript with the expected size of 881 bp (MRC-5, Fig. 1Citation ). A second, minor band of ~553 bp was also present. As reported previously, the decorin gene may be expressed in the form of two species of RNA ~0.5 kb apart in size (30) . When we examined two ovarian cancer cell lines for decorin expression (SKOV3 and 2774), we found expression of the decorin gene in both cell lines, shown again by the presence of two bands of product (Fig. 1)Citation . These same two transcription products were also seen in cDNA made from extracts of fresh ovarian carcinoma (epithelial ovarian cancer, Fig. 1Citation ). We also saw decorin expression in the SW480 colon cancer cell line. Colon cancer cells were previously reported to be negative for decorin transcripts when the less sensitive Northern blot assay was used (10) . Interestingly, immortalized normal ovarian epithelial cells (kindly provided by Rosemarie Schmandt, The University of Texas M. D. Anderson Cancer) also expressed both decorin transcripts (immortalized ovarian epithelial cells, Fig. 1Citation ). It is obvious that the decorin transcripts from the fibroblasts were in much greater abundance because we used up to five times more RNA (1.1–4.35 µg) in the cDNA reactions from the epithelial cells than was used in the cDNA reactions from the fibroblasts (0.85 µg). Also, the relationship between the amount of product and input RNA in RT-PCR reactions is logarithmic, not linear (28 , 31) . However, it is clear that epithelial cells, including tumor cells, do produce decorin transcripts. Also, in contrast to fibroblasts, the epithelial cells show an equal or greater expression of the smaller decorin transcript, perhaps by using an alternate splicing site that had been described previously (32) .



View larger version (46K):
[in this window]
[in a new window]
  		Fig. 1. Detection of decorin transcripts in ovarian cancer cells. RNA extracted from the indicated cells was reverse transcribed with Moloney virus reverse transcriptase and amplified with Taq polymerase using primers specific for decorin. The amplified DNA was displayed on a 1.6% agarose gel, stained with ethidium bromide, and photographed under UV light. A molecular weight ladder of DNA fragments of indicated bp was used to demonstrate the size of the amplified products. The lanes are as follows: M.W., molecular weight markers; MRC-5, fibroblasts; IOE, immortalized ovarian epithelial cells; EOC, epithelial ovarian cancer; SKOV3 and 2774, ovarian tumor cells; and SW480, colon tumor cells. Arrows point to bands at 881 and 553 bp.

 
Immunostaining of Ovarian Cancer Specimens for Decorin.
Because we could not find any previous work describing decorin expression in ovarian cancer cells, and we had found transcripts for decorin in both ovarian cancer cell lines and tumors, we thought it possible that this type of tumor may actually express decorin. To examine this possibility, frozen sections of papillary serous ovarian carcinomas were stained for decorin as described in "Materials and Methods." Eighteen tumor specimens were examined using a peptide-specific antidecorin polyclonal antibody (kindly provided by Telios/Integra), and all of the specimens showed intense staining of the tumor stroma. An example is shown in Fig. 2ACitation . Similar results were obtained when a decorin-specific monoclonal antibody was used to stain a representative section shown in Fig. 2BCitation . Both examples clearly show that whereas the peritumoral stroma stained heavily for decorin, the tumor cells did not. This result is in agreement with previous findings in other tumors (10 , 33) . Thus, although ovarian cancer cells express decorin transcripts, they did not appear to produce significant amounts of protein.



View larger version (95K):
[in this window]
[in a new window]
  		Fig. 2. Histochemical staining of human ovarian tumors with antidecorin antibody. A 5-µm section of a frozen tumor was stained for decorin expression as described in "Materials and Methods." In A, a section was stained with polyclonal antibody (from Telios/Integra), and in B, a section was stained with monoclonal antibody.

 
We were also unable to detect decorin in ethanol/potassium acetate precipitates of conditioned medium from cultured ovarian cancer cells (data not shown). We next decided to see whether decorin was produced in tumor cells but destroyed before being released from the cell. Thus, we examined cells treated with a proteasome inhibitor to see whether we would then detect decorin protein in treated cells. This prevents the movement of unbiquitinated protein into protostomes and results in the buildup of ubiquitin molecules on protein, thus forming distinctive ladders when examined by Western blot analysis.

Ovarian Cancer Cells Treated with the Proteasome Inhibitor MG132.
Ovarian cancer cells were cultured on an 8-well chambered microscope slide and treated overnight with increasing concentrations (25–100 mM) of MG132. The cells were then stained for decorin. As shown in Fig. 3Citation , cells treated with 25 µM (and 50 µM, data not shown) MG132 exhibited a pronounced increase in staining for decorin. The pattern of staining was almost exclusively perinuclear. For this reason, the nuclear counterstain ordinarily used was not applied. The MG132-induced increase in staining was much greater in the 2774 cell line. The dose of 100 µM MG132 was toxic to both cell lines but was better tolerated by the 2774 cell line (data not shown).



View larger version (97K):
[in this window]
[in a new window]
  		Fig. 3. Histochemical staining for decorin in ovarian cancer cells treated with a proteasome inhibitor. SKOV3 and 2774 ovarian cancer cells were grown on a microscope slide and treated overnight with 50 µM MG132. The cells were fixed in acetone at room temperature and stained as for decorin as described in "Materials and Methods." x600.

 
MG132-treated cells were also analyzed by Western blot analysis. As seen in Fig. 4Citation , extracts from both cell lines exhibited a protein ladder detected with decorin-specific monoclonal antibody. Treating cells with MG132 obviously resulted in considerably increased production of these antidecorin-stained proteins, which is easily seen when the blot of the housekeeping gene GAPDH is used to compare sample loading. Such ladders are commonly produced by ubiquitination. Also, ubiquitin complexes have been shown by immunofluorescent staining to be perinuclear aggregates (34) , which tallies with the perinuclear staining for decorin we saw in Fig. 3Citation .



View larger version (69K):
[in this window]
[in a new window]
  		Fig. 4. Western blot detection of decorin protein in human cancer cells treated with a proteasome inhibitor. A, T-25 flasks of SKOV3 or 2774 ovarian cancer cells were treated overnight with 25 µM MG132, lysed in SDS sample buffer, boiled for 5 min, separated on a 10% polyacrylamide gel, blotted onto nitrocellulose, and probed with mouse antidecorin IgG (top panel) or mouse anti-GAPDH monoclonal antibody (bottom panel). The blot was developed by treatment with HRP-conjugated antimouse IgG, soaked in luminol, and then exposed on X-ray film. B, a longer exposure of the decorin blot is shown.

 
Detection of 6-Sulfate Linkage in Ovarian Tumor Stromal Decorin.
The stroma of colon tumors contains high levels of 6-chondroitin sulfate (10) , and peritoneal tumors of colon and ovarian cancer commonly are highly fibrotic. Thus, it occurred to us that cells common to fibrotic tissue may be the source of the type of decorin found in tumor stroma. To investigate this possibility, serial sections of two ovarian tumors were stained with antibody for 6-chondroitin sulfate and for smooth muscle {alpha}-actin, a molecule characteristic of myofibroblasts (20) , cells commonly associated with fibrosis (24) . As demonstrated in Fig. 5, A and BCitation , heavy staining by both antibodies was colocalized in the tumor stroma of both specimens. We next sought to determine the specificity of the role played by the tumor in expression of 6-chondroitin sulfate by stromal myofibroblasts.



View larger version (70K):
[in this window]
[in a new window]
  		Fig. 5. Histochemical staining of human ovarian tissue with anti-6-sulfate chondroitin or anti-smooth muscle {alpha}-actin antibody. Serial 5-µm sections of paraffin-embedded tumors (A and B) or endometrium (C and D) were stained with anti-6-sulfate chondroitin (A and C) or anti-smooth muscle {alpha}-actin antibody (B and D) as described in "Materials and Methods." Arrows point to regions of staining overlap.

 
Endometriosis is a noncancerous gynecological condition that may be associated with fibrosis. Serial sections were made of tissue obtained from two ovaries taken from patients who had undergone oophorectomy for the treatment of their endometriosis and stained as described in Fig. 5, A and BCitation . As in the tumor specimens shown above, we saw overlapping of staining by smooth muscle {alpha}-actin and by 6-sulfate chondroitin (Fig. 5, C and D)Citation . These data support our hypothesis that 6-sulfate chondroitin is a natural product of myofibroblasts and that the presence of this form of chondroitin sulfate in tumor stroma could be independent of tumor cell regulation of its manufacture.


   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
It seems likely that tumor cells would acquire protective mechanisms against the effects of decorin because of the many ways in which decorin inhibits tumor growth. These include binding to thrombospondin-1 and blocking the spread and growth of metastatic tumor cells (17 , 35 , 36) , inhibiting cell proliferation via up-regulation of expression of the p21Waf1/Cip1 molecule in various types of tumor cells (5) including ovarian cancer cells (4) , and binding to and inhibiting activated TGF-ß molecules (15) . It was shown previously that decorin was not expressed in colon cancer (10) . Similarly, decorin production was seen to be inhibited in guinea pig bile duct carcinoma (33) . As we have shown, tumor cells appear to be inhibited from producing decorin through a posttranscriptional mechanism. Although we detected the presence of decorin transcripts in ovarian tissue and cultured cells (and colon tumor cells), we were unable to detect any exported decorin. We have developed, in this study, preliminary evidence showing that decorin may be cotranslationally ubiquitinated and destroyed in ovarian cancer cells. This same fate was shown to occur for the core protein of mutant forms of aggrecan, another proteoglycan (37) .

Decorin is also modified in tumor stromal cells (10) . This modification may act to protect the tumor from the growth-inhibitory effects of decorin. The high proportion of chondroitin sulfate side chains found in tumor-associated decorin (10 , 33) may allow the tumor to grow more aggressively because chondroitin sulfate side chains are 20-fold less effective in retarding tumor cell migration than dermatan sulfate side chains (13) . A question we set out to answer in this study was how the tumor directs the stroma to produce a decorin component of the cell matrix so favorable to tumor cell growth. It was shown in colon cancer that the decorin gene of stromal cells was hypomethylated, thus facilitating the production of decorin (10) . However, it was not shown how the tumor could regulate this activity. We questioned whether the tumor acted, instead, to attract tumor site cells predisposed to produce a stromal matrix favorable to tumor growth. Because many tumors have highly fibrotic stroma, we were particularly interested in myofibroblasts, which are a common feature in many forms of fibrosis. We showed that tumor stroma contains very high expression of smooth muscle {alpha}-actin, a signature protein for myofibroblasts, and that staining for smooth muscle {alpha}-actin largely overlapped with staining for 6-chondroitin sulfate, a form of chondroitin sulfate found in high concentrations in tumor stroma (10) . We showed that fibrotic ovarian tissue from patients with endometriosis also stained with smooth muscle {alpha}-actin and 6-chondroitin sulfate IgGs. Thus, we concluded that myofibroblasts are the probable source of the 6-chondroitin sulfate associated with tumor stroma and that they may produce it independently of tumor coordination. This seems like a reasonable proposal because many types of tumors, including tumors of the ovary (21 , 22) , produce significant levels of TGF-ß, which could behave as a chemotactic agent for myofibroblasts (24) . GM-CSF, also produced by ovarian cancer cells (23) , has also been implicated in myofibroblast accumulation (20 , 24) . However, it also remains possible that epithelial cells, including tumor cells, interact with myofibroblasts after they have accumulated to induce them to produce this form of chondroitin sulfate. This might be affected by the high levels of TGF-ß involved in causing them to accumulate (20 , 24) . Interestingly, another protective role, i.e., to sequester the tumor and thus protect it from immune cells, has also been proposed recently for stromal myofibroblasts (38) .

Because we see similar transcript expression in epithelial cells and tumors, it is interesting to speculate that, compared with decorin transcripts found in fibroblasts, the severely reduced level of transcription and the shift in emphasis toward the smaller transcript may reflect a genomic event that occurs during epithelial cell development. The decorin gene is located at 12q23 (32) , which, coincidentally, is a common site for LOH in several types of tumors, including ovarian, gastric, and pancreatic cancers (39) . LOH at this locus has been observed in 30% of the ovarian tumors examined (40) . A LOH could suggest the presence of a tumor suppressor gene, and although two other potential tumor suppressor genes (i.e., TEL and p27Kip) are both located at that site, neither was shown to be altered in ovarian tumors with LOH at 12q23 (40) . On the other hand, a competent decorin gene, when transferred into tumor cells, was shown to reduce or abrogate tumorigenicity by the transformed cells (2 , 3) . Recent microarray studies have reported that decorin gene expression is significantly reduced in endometrial (41) and ovarian (42) tumor tissues. Our RT-PCR data showed that epithelial cells almost exclusively express the transcript that probably results from the use of an alternative splicing site that fails to make functional decorin. Thus, decorin may represent an example of a potential tumor suppressor gene that becomes inactivated in epithelial cells as a result of normal cell differentiation.


   ACKNOWLEDGMENTS
 
We thank Rebecca Patenia for her histochemical staining contribution to this work.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 To whom requests for reprints should be addressed, at Department of Gynecologic Oncology, The University of Texas M. D. Anderson Cancer Center (440), 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-2764; Fax: (713) 792-7586; E-mail: rfreedma{at}mdanderson.org Back

2 The abbreviations used are: TGF-ß, transforming growth factor ß; GM-CSF, granulocyte/macrophage colony-stimulating factor; HRP, horseradish peroxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT-PCR, reverse transcription-PCR; LOH, loss of heterozygosity. Back

Received 9/11/01; revised 3/ 8/02; accepted 3/18/02.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Prydz K., Dalen K. T. Synthesis and sorting of proteoglycans. J. Cell Sci., 113: 193-205, 2000.[Abstract]
  2. Santra M., Skorski T., Calabretta B., Lattime E. C., Iozzo R. V. De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells. Proc. Natl. Acad. Sci. USA, 92: 7016-7020, 1995.[Abstract/Free Full Text]
  3. Ständer M., Naumann U., Dumitrescu L., Heneka M., Loschmann P., Gulbins E., Dichgans J., Weller M. Decorin gene transfer-mediated suppression of TGF-ß synthesis abrogates experimental malignant glioma growth in vivo. Gene Ther., 5: 1187-1194, 1998.[CrossRef][Medline]
  4. Nash M. A., Loercher A. E., Freedman R. S. In vitro growth inhibition of ovarian cancer cells by decorin: synergism of action between decorin and carboplatin. Cancer Res., 59: 6192-6196, 1999.[Abstract/Free Full Text]
  5. Santra M., Mann D. M., Mercer E. W., Skorski T., Calabretta B., Iozzo R. V. Ectopic expression of decorin protein core causes a generalized growth suppression in neoplastic cells of various histogenetic origin and requires endogenous p21, an inhibitor of cyclin-dependent kinases. J. Clin. Investig., 100: 149-157, 1997.[Medline]
  6. De Luca A., Santra M., Baldi A., Giordano A., Iozzo R. V. Decorin-induced growth suppression is associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases. J. Biol. Chem., 271: 18961-18965, 1996.[Abstract/Free Full Text]
  7. Iozzo R. V., Moscatello D. K., McQuillan D. J., Eichstetter I. Decorin is a biological ligand for the epidermal growth factor receptor. J. Biol. Chem., 274: 4489-4492, 1999.[Abstract/Free Full Text]
  8. Patel S., Santra M., McQuillan D. J., Iozzo R. V., Thomas A. P. Decorin activates the epidermal growth factor receptor and elevates cytosolic Ca2+ in A431 carcinoma cells. J. Biol. Chem., 273: 3121-3124, 1998.[Abstract/Free Full Text]
  9. Moscatello D. K., Santra M., Mann D. M., McQuillan D. J., Wong A. J., Iozzo R. V. Decorin suppresses tumor cell growth by activating the epidermal growth factor receptor. J. Clin. Investig., 101: 406-412, 1998.[Medline]
  10. Adany R., Heimer R., Caterson B., Sorrell J. M., Iozzo R. V. Altered expression of chondroitin sulfate proteoglycan in the stroma of human colon carcinoma. Hypomethylation of PG-40 gene correlates with increased PG-40 content and mRNA levels. J. Biol. Chem., 265: 11389-11396, 1990.[Abstract/Free Full Text]
  11. Brown L. F., Guidi A. J., Schnitt S. J., Van De Water L., Iruela-Arispe M. L., Yeo T. K., Tognazzi K., Dvorak H. F. Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast. Clin. Cancer Res., 5: 1041-1056, 1999.[Abstract/Free Full Text]
  12. Harada T., Shinohara M., Nakamura S., Oka M. An immunohistochemical study of the extracellular matrix in oral squamous cell carcinoma and its association with invasive and metastatic potential. Virchows Arch., 424: 257-266, 1994.[Medline]
  13. Merle B., Durussel L., Delmas P. D., Clecardin P. Decorin inhibits cell migration through a process requiring its glycosaminoglycan side chain. J. Cell. Biochem., 75: 538-546, 1999.[CrossRef][Medline]
  14. Yamaguchi Y., Mann D. M., Ruoslahti E. Negative regulation of transforming growth factor ß by the proteoglycan decorin. Nature (Lond.), 346: 281-284, 1990.[CrossRef][Medline]
  15. Hildebrand A., Romaris M., Rasmussen L. M., Heinegard D., Twardzik D. R., Border W. A., Ruoslahti E. Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor ß. Biochem. J., 302: 527-534, 1994.
  16. Teicher B. A., Maehara Y., Kakeji Y., Ara G., Keyes S. R., Wong J., Herbst R. Reversal of in vivo drug resistance by the transforming growth factor ß inhibitor decorin. Int. J. Cancer, 71: 49-58, 1997.[CrossRef][Medline]
  17. Merle B., Malaval L., Lawler J., Delmas P., Clezardin P. Decorin inhibits cell attachment to thrombospondin-1 by binding to a KKTR-dependent cell adhesive site present within the N-terminal domain of thrombospondin-1. J. Cell. Biochem., 67: 75-83, 1997.[CrossRef][Medline]
  18. Takeuchi Y., Kodama Y., Matsumoto T. Bone matrix decorin binds transforming growth factor ß and enhances its bioactivity. J. Biol. Chem., 269: 32634-32638, 1994.[Abstract/Free Full Text]
  19. Miura S., Kodaira S., Hosoda Y. Immunohistologic analysis of the extracellular matrix components of the fibrous stroma of human colon cancer. J. Surg. Oncol., 53: 36-42, 1993.[Medline]
  20. Rubbia-Brandt L., Sappino A. P., Gabbiani G. Locally applied GM-CSF induces the accumulation of {alpha}-smooth muscle actin-containing myofibroblasts. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol., 60: 73-82, 1991.[Medline]
  21. Gordinier M., Zhang H. Z., Kidd L., Patenia R., Levy L., Katz R. L., Nash M. A., Edwards C. L., Platsoucas C. D., Freedman R. S. Quantitative analysis of transforming growth factor ß1 and ß2 on ovarian cancer. Clin. Cancer Res., 5: 2498-2505, 1999.[Abstract/Free Full Text]
  22. Henriksen R., Gobl A., Wilander E., Berg K., Miyazono K., Funa K. Expression and prognostic significance of TGF-ß isotypes, latent TGF-ß1 binding protein, TGF-ß type I and type II receptors, and endoglin in normal ovary and ovarian neoplasms. Lab. Investig., 73: 213-220, 1995.[Medline]
  23. Merogi A. J., Marrogi A. J., Ramesh R., Robinson W. R., Fermin C. D., Freeman S. M. Tumor-host interaction: analysis of cytokines, growth factors, and tumor-infiltrating lymphocytes in ovarian carcinomas. Hum. Pathol., 28: 321-331, 1997.[CrossRef][Medline]
  24. Xing Z., Tremblay G. M., Sime P. J., Gauldie J. Overexpression of granulocyte-macrophage colony-stimulating factor induces pulmonary granulation tissue formation and fibrosis by induction of transforming growth factor ß1 and myofibroblast accumulation. Am. J. Pathol., 150: 59-66, 1997.[Abstract]
  25. Stokes M. B., Hudkins K. L., Zaharia V., Taneda S., Alpers C. E. Up-regulation of extracellular matrix proteoglycans and collagen type I in human crescentic glomerulonephritis. Kidney Int., 59: 532-542, 2001.[CrossRef][Medline]
  26. Xie W. Q., Rothblum L. I. Rapid, small-scale RNA isolation from tissue culture cells. Biotechniques, 11: 324-, 326327, 1991.[Medline]
  27. Sambrook J., Fritsch E. F., Maniatis T. . Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY 1989.
  28. Nash M. A., Lenzi R., Platsoucas C. D., Freedman R. S. RT-PCR quantitation of cytokine responses in vivo from specimens containing small numbers of cells during bioimmunotherapy. J. Immunol. Methods, 219: 169-179, 1998.[CrossRef][Medline]
  29. Santosm A. N., Kehlen A., Schutte W., Langner J., Riemann D. Regulation by transforming growth factor ß1 of class II mRNA and protein expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis. Int. Immunol., 10: 601-607, 1998.[Abstract/Free Full Text]
  30. Krusius T., Ruoslahti E. Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc. Natl. Acad. Sci. USA, 83: 7683-7687, 1986.[Abstract/Free Full Text]
  31. Kotake S., Schumacher H. R., Jr., Wilder R. L. A simple nested RT-PCR method for quantitation of the relative amounts of multiple cytokine mRNAs in small tissue samples. J. Immunol. Methods, 199: 193-203, 1996.[CrossRef][Medline]
  32. Danielson K. G., Fazzio A., Cohen I., Cannizzaro L. A., Eichstetter I., Iozzo R. V. The human decorin gene: intron-exon organization, discovery of two alternatively spliced exons in the 5' untranslated region, and mapping of the gene to chromosome 12q23. Genomics, 15: 146-160, 1993.[CrossRef][Medline]
  33. Yeo T-K., Brown L., Dvorak H. F. Alterations in proteoglycan synthesis common to healing wounds and tumors. Am. J. Pathol., 138: 1437-1450, 1991.[Abstract]
  34. Wojcik C., Schroeter D., Wilk S., Lamprecht J., Paweletz N. Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome. Eur. J. Cell Biol., 71: 311-318, 1996.[Medline]
  35. Roberts D. D. Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J., 10: 1183-1191, 1996.[Abstract]
  36. Wang T. N., Qian X., Granick M. S., Solomon M. P., Rothman V. L., Berger D. H., Tuszynski G. P. Thrombospondin-1 (TSP-1) promotes the invasive properties of human breast cancer. J. Surg. Res., 63: 39-43, 1996.[CrossRef][Medline]
  37. Domowicz M. S., Pirok E. W., III, Novak T. E., Schwartz N. B. Role of the C-terminal G3 domain in sorting and secretion of aggrecan core protein and ubiquitin-mediated degradation of accumulated mutant precursors. J. Biol. Chem., 275: 35098-35105, 2000.[Abstract/Free Full Text]
  38. Lieubeau B., Heymann M. F., Henry F., Barbieux I., Meflah K., Gregoire M. Immunomodulatory effects of tumor-associated fibroblasts in colorectal-tumor development. Int. J. Cancer, 81: 629-636, 1999.[CrossRef][Medline]
  39. Yang X. H., Huang S. PHM1 (PRDM4), a new member of the PR-domain family, maps to a suppressor locus on human chromosome 12q23–q24.1. Genomics, 61: 319-325, 1999.[CrossRef][Medline]
  40. Hatta Y., Takeuchi S., Yokota J., Koeffler H. P. Ovarian cancer has frequent loss of heterozygosity at chromosome 12p12.3–13.1 (region of TEL and Kip1 loci) and chromosome 12q23-ter: evidence for two new tumour-suppressor genes. Br. J. Cancer, 75: 1256-1262, 1997.[Medline]
  41. Smid-Koopman E., Blok L. J., Chadha-Ajwani S., Helmerhorst T. J. M., Brinkmann A. O., Huikeshoven F. J. Gene expression profiles of human endometrial cancer samples using a cDNA-expression array technique: assessment of an analysis method. Br. J. Cancer, 83: 246-251, 2000.[CrossRef][Medline]
  42. Shridhar V., Lee J., Pandita A., Iturria S., Avula R., Staub J., Morrissey M., Calhoun E., Sen A., Kalli K., Keeney G., Roche P., Cliby W., Lu K., Schmandt R., Mills G. B., Bast R. C., James C. D., Couch F. J., Hartmann L. C., Lillie J., Smith D. I. Genetic analysis of early- versus late-stage ovarian tumors. Cancer Res., 61: 5895-5904, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 FASEB J.Home page
M. Magzoub, S. Jin, and A. S. Verkman
Enhanced macromolecule diffusion deep in tumors after enzymatic digestion of extracellular matrix collagen and its associated proteoglycan decorin
FASEB J, January 1, 2008; 22(1): 276 - 284.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
G. B. ten Dam, E. M.A. van de Westerlo, A. Purushothaman, R. V. Stan, J. Bulten, F. C.G.J. Sweep, L. F. Massuger, K. Sugahara, and T. H. van Kuppevelt
Antibody GD3G7 Selected against Embryonic Glycosaminoglycans Defines Chondroitin Sulfate-E Domains Highly Up-Regulated in Ovarian Cancer and Involved in Vascular Endothelial Growth Factor Binding
Am. J. Pathol., October 1, 2007; 171(4): 1324 - 1333.
[Abstract] [Full Text] [PDF]

Home page
 J BiochemHome page
P. Pothacharoen, S. Siriaunkgul, S. Ong-Chai, J. Supabandhu, P. Kumja, C. Wanaphirak, K. Sugahara, T. Hardingham, and P. Kongtawelert
Raised Serum Chondroitin Sulfate Epitope Level in Ovarian Epithelial Cancer
J. Biochem., October 1, 2006; 140(4): 517 - 524.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. G. Seidler, S. Goldoni, C. Agnew, C. Cardi, M. L. Thakur, R. T. Owens, D. J. McQuillan, and R. V. Iozzo
Decorin Protein Core Inhibits in Vivo Cancer Growth and Metabolism by Hindering Epidermal Growth Factor Receptor Function and Triggering Apoptosis via Caspase-3 Activation
J. Biol. Chem., September 8, 2006; 281(36): 26408 - 26418.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J.-X. Zhu, S. Goldoni, G. Bix, R. T. Owens, D. J. McQuillan, C. C. Reed, and R. V. Iozzo
Decorin Evokes Protracted Internalization and Degradation of the Epidermal Growth Factor Receptor via Caveolar Endocytosis
J. Biol. Chem., September 16, 2005; 280(37): 32468 - 32479.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
E. Wang, Y. Ngalame, M. C. Panelli, H. Nguyen-Jackson, M. Deavers, P. Mueller, W. Hu, C. A. Savary, R. Kobayashi, R. S. Freedman, et al.
Peritoneal and Subperitoneal Stroma May Facilitate Regional Spread of Ovarian Cancer
Clin. Cancer Res., January 1, 2005; 11(1): 113 - 122.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
J. Koninger, N. A. Giese, F. F. di Mola, P. Berberat, T. Giese, I. Esposito, M. G. Bachem, M. W. Buchler, and H. Friess
Overexpressed Decorin in Pancreatic Cancer: Potential Tumor Growth Inhibition and Attenuation of Chemotherapeutic Action
Clin. Cancer Res., July 15, 2004; 10(14): 4776 - 4783.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
M. Abe, S. Yamashita, T. Kuramoto, Y. Hirayama, T. Tsukamoto, T. Ohta, M. Tatematsu, M. Ohki, T. Takato, T. Sugimura, et al.
Global expression analysis of N-methyl-N'-nitro-N-nitrosoguanidine-induced rat stomach carcinomas using oligonucleotide microarrays
Carcinogenesis, May 1, 2003; 24(5): 861 - 867.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Santra, C. C. Reed, and R. V. Iozzo
Decorin Binds to a Narrow Region of the Epidermal Growth Factor (EGF) Receptor, Partially Overlapping but Distinct from the EGF-binding Epitope
J. Biol. Chem., September 13, 2002; 277(38): 35671 - 35681.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nash, M. A.
Right arrow Articles by Freedman, R. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nash, M. A.
Right arrow Articles by Freedman, R. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Cell Growth & Differentiation