Coordinate regulation of cyclooxygenase-2 and TGF-ß1 in replication error-positive colon cancer and azoxymethane-induced rat colonic tumors

Jinyi Shao1, Hongmiao Sheng2, Radhika Aramandla1, Michael A. Pereira3, Ronald A. Lubet4, Ernest Hawk4, Liam Grogan4, Ilan R. Kirsch4, M. Kay Washington5, R. Daniel Beauchamp2,6 and Raymond N. DuBois1,6,7,8

1 Departments of Medicine,
2 Surgery,
5 Pathology and
6 Cell Biology, Vanderbilt University Medical Center,
7 Veterans Affairs Medical Center, Nashville, TN 37232,
3 Medical College of Ohio, Toledo, OH 43614 and
4 National Cancer Institute, Bethesda, MD 20892, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Evidence is accumulating which indicates that cyclooxygenase-2 (COX-2) is involved in the pathogenesis of colorectal cancer. We evaluated the expression of COX-2 in replication error-positive (RER) colon cancers, colon cancers metastatic to liver and azoxymethane (AOM)-induced rat colonic tumors. Immunohistochemistry showed that COX-2 was low to undetectable in normal human mucosa, but abundant in the RER adenocarcinomas we examined. COX-2 immunoreactivity in metastatic colon cancers was less abundant, but clearly detectable. In the colon of AOM-treated rats, COX-2 protein was not detectable in normal mucosa, but present in most of the epithelial cells comprising the tumors. The TGF-ß1 staining pattern in these human and rat tumors was similar to that observed for COX-2. The role of TGF-ß in RER adenocarcinomas is complex because of the increased mutation rate of TGF-ß type II receptors. Northern analysis showed abundant TGF-ß1 mRNA in AOM-induced tumors, but not in paired mucosa. TGF-ß1 induced the expression of COX-2 mRNA and protein in intestinal epithelial cells (IEC-6). Chronic TGF-ß1 treatment caused a TGF-ß-dependent overexpression of COX-2 in rat intestinal epithelial cells (RIE-1). TGF-ß1 may regulate COX-2 expression during the colonic adenoma to carcinoma sequence.

Abbreviations: AOM, azoxymethane; COX, cyclooxygenase; HNPCC, non-polyposis colorectal cancer; NSAID, non-steroidal anti-inflammatory drug; PBS, phosphate-buffered saline; RER, replication error-positive; TGF-ß, transforming growth factor ß.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Numerous studies have demonstrated a 40–50% reduction in relative risk for colorectal cancer in individuals taking non-steroidal anti-inflammatory drugs (NSAIDs) compared with those not taking these agents (16). Individuals with familial adenomatous polyposis who take sulindac develop a 35–55% reduction in adenoma size and number (711). In the azoxymethane (AOM)-induced colorectal carcinogenesis model, cyclooxygenase inhibitors exhibit chemoprotective effects as judged by a reduction in the frequency and number of premalignant and malignant lesions (1214). Reddy et al. have demonstrated a marked reduction in aberrant crypt formation and tumor burden in rats by treatment with a highly selective cyclooxygenase (COX)-2 inhibitor (15,16).

We have previously reported increased COX-2 expression in human colorectal adenocarcinomas when compared with normal adjacent colonic mucosa (17). These findings have been confirmed by other investigators who have shown elevated levels of COX-2 protein in colorectal tumors by western blotting (18) and immunohistochemical staining (19). We have also observed markedly elevated levels of COX-2 mRNA and protein in colonic tumors that develop in rodents following carcinogen treatment (20) and in adenomas taken from Min mice (21). A recent report by Oshima et al. indicates that COX-2 may play an extremely important role in the development of adenomas following loss of APC function (22,23). Adenomas from APC{Delta}716 mice were found to have elevated COX-2 levels, although the precise cellular localization of the COX-2 protein was not defined by immunostaining. Additionally, treatment of APC{Delta}716 mice with a highly selective COX-2 inhibitor significantly reduces tumor multiplicity. Taken together, these results provide strong genetic and pharmacological evidence for a role of COX-2 in adenoma formation following loss of APC function.

Although overexpression of COX-2 in human and rodent intestinal tumors is widely observed, the mechanisms responsible for expression of COX-2 in colonic tumors are not understood. The transforming growth factors ß (TGF-ßs) are 25 kDa homodimeric polypeptides belonging to a superfamily of growth regulatory molecules. TGF-ßs are known to regulate many biological processes, including cellular proliferation, cell migration, differentiation and extracellular matrix deposition (2426). TGF-ß has previously been characterized as a potent growth inhibitor for cultured rat intestinal crypt cells (2730). Although loss of sensitivity to TGF-ß growth inhibitory effects may be a key step in the escape of intestinal epithelial tumors from normal growth control, the exact role of TGF-ß in neoplastic transformation of intestinal epithelium is still unclear. There is mounting evidence that TGF-ß may enhance malignant transformation and tumor progression of several different epithelial tumors under certain circumstances (3136). One of the remarkable effects of TGF-ß on intestinal epithelial cells and other cell types is the induction or augmentation of COX-2 expression (3741). TGF-ß treatment induces COX-2 expression at both the mRNA and protein levels in rat intestinal epithelial cells (RIE-1) (41). While TGF-ß expression is normally restricted to the lumenal third of the colonic epithelium, in colonic epithelium immediately adjacent to carcinomas TGF-ß was found to be diffusely distributed throughout the colonic glands. Abundant TGF-ß1 immunostaining was present in 46 of 48 cancers and nine of 10 adenomas (42). Based upon the observations in human colon cancer that COX-2 was overexpressed in 85–90% of human colon cancers (17) and that TGF-ß was abnormally expressed in >90% of human colon cancers (42), we hypothesized that TGF-ß may play a role in the regulation of COX-2 expression during the adenoma to carcinoma sequence of events involved in the neoplastic transformation of colonic epithelial cells.

This study describes the expression pattern of COX-2 in replication error-positive (RER) colon cancers, in metastatic colon carcinomas and in AOM-induced rat colonic tumors. We also investigated the concurrent expression of TGF-ß in these human and rat tumors and confirmed that TGF-ß induces COX-2 expression and that chronic treatment with TGF-ß1 causes constitutive expression of COX-2 in rat intestinal epithelial cells. TGF-ß1 may regulate COX-2 expression during the colonic adenoma to carcinoma sequence of events under certain circumstances.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Patient samples
Ten RER colon cancers were obtained at the National Cancer Institute as previously described (43). An RER-positive phenotype was defined by instability that precedes transformation and involves widespread alterations in multiple microsatellite markers. The patients described in this study include six hereditary non-polyposis colorectal cancer (HNPCC) patients who met the Amsterdam/Bethesda criteria for HNPCC. Three patients have been proven to carry germline mutations of hMSH2 or hMLH1. The germline status in one patient is not clear.

Formalin-fixed and paraffin-embedded tissue blocks of liver metastatic colonic adenocarcinomas from eight patients were obtained under a protocol approved by the Vanderbilt University Medical Center Committee for Protection of Human Subjects. These specimens were then sectioned and processed as described below for histological analysis.

Cell culture
Rat intestinal epithelial cells (IEC-6 and RIE-1) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone Laboratories, Logan, UT). Aliquots of 3 ng/ml of either porcine platelet TGF-ß1 or human recombinant TGF-ß1 (R & D Systems, Minneapolis, MN) were used for cell treatment. For chronic TGF-ß1 treatment, medium containing 3 ng/ml fresh TGF-ß1 was replaced every 3 days.

Carcinogen treatment, tissue procurement and RNA isolation
The experimental design and protocols have been described previously (20). Seven-week-old male Fisher 344 rats were administered AOM (30 mg/kg). Colonic tumors and normal tissues were obtained 46 weeks later. In each case, accompanying normal mucosa from the same animal was collected for comparison. All tissues were either fixed overnight in formaldehyde, then transfered to 70% alcohol and stored at 4°C for immunohistochemistry or placed in cryovials, flash frozen in liquid nitrogen and stored at –80°C. Total RNA and protein lysates were isolated from these samples using previously reported methods (20).

Northern blot analysis
RNA samples (20 µg/lane) were separated on formaldehyde–agarose gels and blotted onto nitrocellulose membranes. The blots were hybridized with cDNA probes labeled with [{alpha}-32P]dCTP by random primer extension. After hybridization and washes, the blots were exposed to X-ray film for autoradiography. 18S rRNA signals were used as internal controls to determine the integrity of the RNA and the equality of loading among lanes. The results were semi-quantitatively analyzed using NIH image optical scanning and densitometric software.

Western blotting analysis
Immunoblot analysis was performed as previously described (30). Briefly, the cells were lysed for 30 min in RIPA buffer [1x phosphate-buffered saline (PBS), 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 mM sodium orthovanadate], then the clarified cell lysates were denatured and fractionated by SDS–PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membrane. The filters were then probed with the indicated antibodies, developed by enhanced chemiluminescence (Amersham, Arlington Heights, IL) and exposed to XAR5 film (Kodak, Rochester, NY). Quantitation was determined by densitometry. All antibodies used for this study were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Immunohistochemical staining
For immunostaining, paraffin-embedded sections (5 µm) were deparaffinized and rehydrated in xylene, graded alcohol and PBS, respectively. Endogenous peroxidase activity was quenched by incubating the sections in 0.3% hydrogen peroxide for 20 min at room temperature. After the sections were blocked in 10% normal rabbit serum in PBS (goat serum for TGF-ß1 staining) for 1 h primary antibodies were applied to the sections and incubated overnight at 4°C. The sections were then incubated with biotinylated rabbit anti-goat antibody (goat anti-rabbit for TGF-ß1 staining) and ABC-AP reagent according to the manufacturer's instructions (Vectorstain ABC-AP kit; Vector Laboratories, Burlingame, CA). Peroxidase activity was shown by applying 3,3'-diaminobenzidine tetrahydrochloride containing 0.02% hydrogen peroxide for 10 min. The sections were counterstained with toludine blue O and mounted with coverslips. To block the activity of antibody, a 10-fold amount of blocking peptide was incubated with the antibody at 4°C for 4 h prior to application to the sections. Goat polyclonal anti-COX-1 antibody (C-20), goat polyclonal anti-COX-2 antibody (N-20) and rabbit polyclonal anti-TGF-ß1(V) antibody and COX-1 and COX-2 blocking peptides were purchased from Santa Cruz Biotechnology.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Expression of COX-1, COX-2 and TGF-ß1 in human tumors
COX-1 and COX-2 share ~70% sequence identity and are highly homologous in amino acid sequence. In order to avoid cross-reactivity between the antibodies we tested anti-COX-1 and anti-COX-2 antibodies from several sources and confirmed that the antibodies used in this study were relatively specific and exhibited undetectable cross-reactivity between COX-1 and COX-2 proteins. As demonstrated in Figure 1Go, the anti-COX-1 antibody (C-20) detected only the recombinant COX-1 protein and the anti-COX-2 antibody (N-20) detected the COX-2 recombinant protein and COX-2 in the cell lysate from a COX-2-positive human colon cancer cell line (HCA-7) (44).



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Fig. 1. Analysis of the specificity of anti-COX-1 (C-20) and anti-COX-2 (N-20) antibodies. Recombinant COX-1 peptide (1 µg), 20 µg of HCA-7 lysate and 1 µg of recombinant COX-2 peptide were separated by SDS–PAGE, blotted and probed with anti-COX-1 or anti-COX-2 antibody.

 
Immunohistochemical analysis in normal human colon revealed that COX-1 immunoreactivity was present in colonic epithelial cells in the lower third of the crypt. An average of one to three positive cells per crypt was observed with perinuclear localization. The positive cells were evenly dispersed and did not occur in clusters (Figure 2AGo). The expression of COX-1 in RER adenocarcinomas was increased, but only rare tumor cells in an area of mucinous carcinoma were positive. Most positive cells represented the giant cells and macrophages reacting to mucin released by the tumor (Figure 2BGo). Preincubation of anti-COX-1 antibody with COX-1 blocking peptide completely abolished the positive staining in a serial section of the same tumor (Figure 2CGo). Immunostaining for COX-2 showed that the surface colonocytes of normal mucosa were positive, with a granular cytoplasmic staining pattern (Figure 2DGo). Positive COX-2 immunoreactivity was detected in 10 of 10 RER tumors. The tumor cells exhibited diffusely and strongly positive COX-2 immunoreactivity with supranuclear cytoplasmic staining. Perinuclear staining was observed in some tumor cells (Figure 2E and FGo). Preincubation of anti-COX-2 antibody with COX-2 peptide blocked the detection of COX-2 in a section of the same tumor (Figure 2GGo). Blocking the COX-2 antibody with COX-1 peptide did not alter the staining pattern, but slightly reduced the intensity as compared with the results with untreated COX-2 antibody (Figure 2HGo). It was previously reported that expression of TGF-ß1, 2 and 3 is restricted to the non-proliferative differentiated epithelial compartments nearer the lumenal surface in both the small and large intestine (28,45). Consistent with these findings, TGF-ß1 immunoreactivity was localized in the upper third of the crypt in normal colonic mucosa (data not shown). All 10 RER tumors examined in this study exhibited strong positive TGF-ß1 immunoreactivity. Tumor cells showed diffuse fine cytoplasmic staining, with a superimposed Golgi-type staining pattern (Figure 2IGo).



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Fig. 2. Immunostaining for COX-1, COX-2 and TGF-ß1 in RER colon cancer and liver metastatic colonic carcinomas. Tumors and normal mucosa were fixed in 10% formaldehyde. The paraffin-embedded sections were subjected to immunohistochemical staining for COX-1, COX-2 and TGF-ß1 as described in Materials and methods. (A) COX-1 immunoreactivity in normal colonic mucosa (original magnification x200). (B) COX-1 immunostaining in RER carcinoma (x200). (C) Blocking COX-1 antibody with COX-1 peptide. The COX-1 antibody was blocked by preincubation of the antibody with a 10-fold amount of COX-1 peptide at 4°C for 4 h. The preincubated antibody was used for immunostaining for detection of COX-1 (x200). (D) COX-2 expression in normal human colonic mucosa (x200). (E and F) COX-2 immunoreactivity in RER carcinoma (x200 and x1000). (G) Blocking COX-2 antibody with COX-2 peptide. The COX-2 antibody was incubated with a 10-fold amount of COX-2 peptide at 4°C for 4 h prior to application to the slide that was prepared from the same tumor used for (E) and (F). (H) Blocking of COX-2 antibody with COX-1 peptide. The COX-2 antibody was incubated with a 10-fold amount of COX-1 peptide at 4°C for 4 h prior to application to the slide (x200). (I) TGF-ß1 expression in RER carcinoma (x1000). (J) COX-1 immunoreactivity in liver metastatic colon cancer (x200). (K) COX-2 expression in liver metastatic colon cancer (x200). (L) Immunostaining for TGF-ß1 in liver metastatic colon cancer (x200).

 
Immunohistochemical staining of colonic carcinomas metastatic to liver showed that COX-1 immunoreactivity was predominantly present in the interstitial cells, but not in the tumor cells (Figure 2JGo). Six out of eight metastatic tumors exhibited positive COX-2 immunoreactivity at a relatively low intensity. The COX-2 protein was predominantly located in the cytoplasm of tumor cells, with a focal supranuclear enhancement staining pattern (Figure 2KGo). Positive staining for TGF-ß1 was observed in eight of eight metastatic tumors and was present in the cytoplasm of tumor cells (Figure 2LGo).

Expression of COX-1, COX-2 and TGF-ß1 in AOM-treated rat colon
We have previously shown that COX-2 protein and mRNA levels are elevated in AOM-induced rat colonic tumors (20). To evaluate the localization of expression, representative tissue sections from neoplastic lesions and normal epithelium were prepared and probed with COX-1 and COX-2 antibodies. COX-1-positive cells were present within the lower and middle portions of the glands in normal mucosa (Figure 3AGo). AOM-induced tumors showed patchy heterogeneous staining for COX-1 protein. COX-1 immunoreactivity was present in the tumor cells, with perinuclear and cytoplasmic staining (Figure 3BGo). Preincubation with COX-1 peptide completely blocked the immunoreactivity of anti-COX-1 antibody (Figure 3CGo). COX-2 immunoreactivity was very low in normal rat colonic epithelium (Figure 3DGo). However, COX-2 expression was detected in some epithelial cells in the mucosa adjacent to AOM-induced adenocarcinomas (data not shown) and was extremely abundant in most of the transformed epithelial cells comprising the adenomas (Figure 3EGo) and adenocarcinomas (Figure 3FGo) from nine of 10 of the tumors examined. As demonstrated in Figure 3GGo, COX-2 immunoreactivity was localized in the perinuclear cytoplasm. Preincubation with COX-2 peptide abolished the immunoreactivity of the anti-COX-2 antibody (Figure 3HGo). COX-1 peptide was unable to block the immunoreactivity of COX-2 antibody (data not shown). The TGF-ß1 staining pattern was similar to that observed for COX-2, being found in adjacent mucosa (Figure 3IGo), in adenomas (Figure 3JGo) and in adenocarcinomas (Figure 3KGo).



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Fig. 3. Immunostaining for COX-1, COX-2 and TGF-ß1 in AOM-treated rat colon. (A) COX-1 expression in normal rat colonic mucosa (original magnification x200). (B) COX-1 immunoreactivity in AOM-induced colonic adenoma (x200). (C) Blocking COX-1 antibody with COX-1 peptide. The COX-1 antibody was incubated with a 10-fold amount of COX-1 peptide at 4°C for 4 h prior to application to the slide which was prepared from the same tumor used for (B). (D) Immunostaining for COX-2 in normal rat mucosa (x200). (E) COX-2 expression in AOM-induced colonic adenoma (x200). (F and G) COX-2 expression in AOM-induced colonic adenocarcinoma (x200 and x1000). (H) Blocking COX-2 antibody with COX-2 peptide. The COX-2 antibody was incubated with a 10-fold amount of COX-2 peptide at 4°C for 4 h prior to application to the slide that was prepared from the same tumor used for (E) and (F). (I) TGF-ß1 immunoreactivity in normal rat colonic mucosa (x200). (J) Expression of TGF-ß1 in AOM-induced colonic adenoma (x200). (K) Expression of TGF-ß1 in AOM-induced colonic adenocarcinoma (x200).

 
In order to confirm the concurrent overexpression of COX-2 and TGF-ß1 in AOM-induced colonic tumors, RNAs from four paired normal mucosae and tumors were subjected to northern analysis for detection of TGF-ß1. As demonstrated in Figure 4Go, low to undetectable levels of TGF-ß1 mRNA were observed in each of the normal mucosa samples (M); however, a TGF-ß1 transcript was apparent in all of the colon tumors examined. The degree of elevation of TGF-ß1 mRNA, as determined by densitometry scanning of the autoradiogram, ranged from 1.5- to 34-fold in the cancer compared with the paired normal mucosa. These results demonstrate that TGF-ß1 RNA levels are increased in colonic tumors that develop in rodents following carcinogen treatment.



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Fig. 4. Northern analysis for TGF-ß1 mRNA in AOM-treated rat colon. Colonic tumor and mucosa were collected from the AOM-treated animals. RNA from paired mucosa (M) and tumor (T) were isolated, separated on a formaldehyde–agarose gel and transferred to nitrocellulose membrane. The levels of TGF-ß1 mRNA were analyzed by northern blotting.

 
Induction of COX-2 by TGF-ß1 treatment
We previously reported that TGF-ß1 treatment induced COX-2 expression at both the mRNA and protein levels in rat intestinal epithelial cells (RIE-1) (41). Here we examined whether TGF-ß1 treatment of rat intestinal epithelial cells (IEC-6) resulted in increased COX-2 expression. Northern blots were prepared and probed with the 32P-labeled cDNAs indicated in Figure 5AGo. The results of this experiment demonstrate that the level of COX-2 mRNA was undetectable in untreated IEC-6 cells. TGF-ß1 treatment resulted in an elevation of the level of COX-2 mRNA starting within 1 h after addition of TGF-ß1. COX-2 mRNA levels increased 9-fold 9 h following TGF-ß1 treatment. The protein extracts from cells treated in an identical manner were evaluated by western blotting to quantitate COX-2 protein levels (Figure 5BGo). We found the COX-2 protein levels increased in a similar temporal fashion to the COX-2 mRNA and were elevated 10-fold 9 h following TGF-ß1 treatment.



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Fig. 5. TGF-ß1 induction of COX-2 in rat intestinal epithelial cells. (A) Induction of COX-2 mRNA by TGF-ß1 treatment in IEC-6 cells. Cells were treated with 3 ng/ml TGF-ß1. RNA was isolated at the indicated time points and subjected to northern analysis for COX-2 expression. (B) Induction of COX-2 protein by TGF-ß1 treatment in IEC-6 cells. Cells were treated with 3 ng/ml TGF-ß1. Total cellular proteins were isolated at the indicated time points. Aliquots of 50 µg of clarified cell lysate from each sample were denatured and fractionated by SDS–PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membrane. The filters were then probed with anti-COX-2 antibody (N-20; Santa Cruz), developed with the ECL system and exposed to X-ray film. (C) The effect of chronic TGF-ß1 treatment on expression of COX-2 in RIE-1 cells. RIE-1 cells were treated with 3 ng/ml TGF-ß1 for 30, 40 and >50 days. Total cellular proteins were isolated at the indicated time points. Aliquots of 50 µg of clarified cell lysate from each sample were analyzed by immunoblotting for detection of the levels of COX-2 protein. P, untreated RIE-1 cells; –20, the cells were treated with TGF-ß1 for >50 days then incubated in medium without TGF-ß1 for another 20 days.

 
In order to determine whether prolonged TGF-ß treatment would cause constitutive expression of COX-2 in intestinal epithelial cells, we treated rat intestinal epithelial (RIE-1) cells with TGF-ß1 for 30, 40 and >50 days. The level of COX-2 protein was evaluated and compared with the level in untreated RIE-1 cells. As shown in Figure 5CGo, the level of COX-2 protein was significantly increased at days 30 and 40 after the cells were treated with TGF-ß1. A 12-fold increase in the level of COX-2 protein was observed after the cells were treated with TGF-ß1 for 50 days. The level of COX-2 protein was significantly reduced when TGF-ß1 was removed from the culture medium. Within 20 days after removal of TGF-ß1, COX-2 immunoreactivity returned to near baseline levels (Figure 5CGo). These results suggest that the overexpression of COX-2 in the RIE-1 cells chronically treated with TGF-ß1 depends upon the continued presence of TGF-ß.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Understanding the molecular events involved in the development of colorectal neoplasia has progressed remarkably during the past decade. Clinical and epidemiological studies have shown a relationship between NSAID use and reduction in relative risk for colorectal adenomas, cancer and colorectal cancer-associated mortality (16). Numerous animal studies have shown a reduction in tumor burden in animals that were treated with a wide variety of NSAIDs following carcinogen treatment (20). Although the mechanism whereby NSAIDs mediate their chemoprotective effects are unknown, inhibition of cyclooxygenase enzymes leading to a reduction in eicosanoid production remains a possibility. We and others have shown that mitogen-inducible cyclooxygenase (COX-2) expression is up-regulated in human colorectal carcinomas (1719). Recent reports have shown a link between the tumorigenic effect of APC mutations and arachidonic acid metabolism by the observation that disruption of the COX-2 gene reduces the number of tumors in mice heterozygous for an APC{Delta}716 mutation by >6-fold (22). The cell type responsible for COX-2 expression in the APC{Delta}716 adenomas is under investigation. Additional evidence supporting a role for COX-2 comes from studies which show a marked reduction in aberrant crypt formation in rodents treated with highly selective COX-2 inhibitors (15,16). In this study, we observed increased expression of COX-2 in RER-positive adenocarcinomas and in colorectal cancers metastatic to the liver. These findings may have potential significance for chemoprevention and chemotherapy in these patients. Our results also demonstrate that COX-2 expression was found predominantly in epithelial cells in colonic carcinomas from both human and carcinogen-treated rat. Neoplastic transformation in the colon can be caused by alterations in a variety of signaling pathways that result from mutations of key genes. We have observed elevated expression of COX-2 in several different model systems for colon cancer, implying the potential importance of COX-2 in colonic carcinogenesis.

The genetic defects underlying RER in colon cancer are mutations of mismatch repair genes, including hMSH2 and hMLH1, hPMS1 and hPMS2. Mutations of mismatch genes is associated with microsatellite instability and subsequent activation of oncogenes and/or inactivation of tumor suppressor genes (46). It has been reported that the TGF-ß type II receptor is mutated within a polyadenine tract in 90% of colorectal cancers with microsatellite instability (47). Seven of 10 patients described in this study exhibit mutated TGF-ß type II receptor (L.Grogan and I.R.Kirsch, unpublished data). Although both TGF-ß type I and II receptors are required for TGF-ß growth inhibition, whether the non-growth related signaling in response to TGF-ß absolutely requires TGF-ß type II receptor remains unclear (48). TGF-ß exhibits a variety of effects which modulate the phenotype of transformed epithelial cells (3236,49). The TGF-ß type II receptor is often defective in transformed epithelial cells. Chen et al. (48) demonstrated in mink lung epithelial cells (Mv1Lu) that overexpression of the dominant negative type II receptor resulted in loss of growth inhibition in response to TGF-ß treatment, however, induction of plasminogen activator inhibitor-1 (PAI-1) and fibronectin proteins and JunB mRNA remained intact. Interestingly, TGF-ß and COX-2 are concurrently overexpressed in the RER tumors examined in this study. Further studies are required to determine whether these observations are of causal significance or simply reflect the phenotypic alteration of RER-positive colon cancer.

One of the rodent models which has been utilized for numerous cancer prevention studies involves the use of AOM-treated Fisher 344 rats. However, the molecular events involved in colorectal carcinogenesis in this rodent model are not completely understood. Obviously, COX-2 is an AOM-targeted gene which enhances cell survival (5052). In this report we found that TGF-ß1 and COX-2 are concurrently overexpressed in the same colonic neoplastic lesions in AOM-treated rats and that chronic TGF-ß treatment resulted in a constitutive overexpression of COX-2 in rat intestinal epithelial (RIE) cells. The increased COX-2 expression in these cells appears to be dependent on treatment with exogenous TGF-ß. These observations have led us to consider the hypothesis that constitutive expression of COX-2 in AOM-induced colonic tumor could be due, in part, to the overexpression of TGF-ß. We recently reported that continuous exposure of non-tumorigenic intestinal epithelial (RIE) cells to TGF-ß1 results in morphological transformation and the acquisition of a tumorigenic phenotype that is associated with up-regulation of COX-2 and down-regulation of the TGF-ß type II receptor (53), implying that COX-2 is a target gene of the TGF-ß signaling pathway and may contribute to TGF-ß-promoted transformation in epithelial cells.

In summary, AOM-induced rat colonic adenoma and adenocarcinoma involve increased expression of COX-2 that is localized in the tumor cells. The elevated expression of COX-2 may result from overexpression of TGF-ß1. However, TGF-ß and COX-2 are also concurrently overexpressed in HNPCC tumors, suggesting more complex relations between TGF-ß and COX-2 in colonic carcinogenesis.


    Acknowledgments
 
This work was supported in part by funds from US Public Health Services grants NIEHS-00267 (R.N.D.), DK-47297 (R.N.D.), CA-69457 and DK-52334 (R.D.B.), CA68485 (Vanderbilt Cancer Center) and NO1-CN-55151-MAO (M.A.P.). R.N.D. is the Mina C.Wallace Professor of Gastroenterology and Cancer Prevention, the recipient of a VA Merit Review Award and an AGA Industry Research Scholar.


    Notes
 
8 To whom correspondence should be addressed at: Department of Medicine/GI, MCN C-2104, Vanderbilt University Medical Center, Nashville, TN 27232-2279, USA Email: duboisrn{at}ctrvax.vanderbilt.edu Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 22, 1998; revised September 24, 1998; accepted October 6, 1998.