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Originally published In Press as doi:10.1074/jbc.M407403200 on November 1, 2004

J. Biol. Chem., Vol. 280, Issue 1, 476-483, January 7, 2005
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Prostaglandin E2 Regulates the Complement Inhibitor CD55/Decay-accelerating Factor in Colorectal Cancer*

Vijaykumar R. Holla{ddagger}, Dingzhi Wang{ddagger}, Joanne R. Brown{ddagger}, Jason R. Mann{ddagger}, Sharada Katkuri{ddagger}, and Raymond N. DuBois{ddagger}§

From the {ddagger}Department of Medicine, Vanderbilt University Medical Center and the §Department of Cancer Biology, Vanderbilt-Ingram Cancer Center, Nashville, Tennessee 37232-2279

Received for publication, July 2, 2004 , and in revised form, September 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase-derived prostaglandin E2 (PGE2) stimulates tumor progression by modulating several proneoplastic pathways. The mechanisms by which PGE2 promotes tumor growth and metastasis through stimulation of cell migration, invasion, and angiogenesis have been fairly well characterized. Much less is known, however, about the molecular mechanisms responsible for the immunosuppressive effects of PGE2. We identified PGE2 target genes and subsequently studied their biologic role in colorectal cancer cells. The complement regulatory protein decay-accelerating factor (DAF or CD55) was induced following PGE2 treatment of LS174T colon cancer cells. Analysis of PGE2-mediated activation of the DAF promoter employing 5'-deletion luciferase constructs suggests that regulation occurs at the transcriptional level via a cyclic AMP/protein kinase A-dependent pathway. Nonsteroidal anti-inflammatory drugs blocked DAF expression in HCA-7 colon cancer cells, which could be restored by the addition of exogenous PGE2. Finally, we observed an increase in DAF expression in the intestinal mucosa of ApcMin+/- mice treated with PGE2 in vivo. In summary, these results indicate a novel immunosuppressive role for PGE2 in the development of colorectal carcinomas.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Immune evasion represents an important mechanism by which cancer cells survive in a hostile environment. This complex process involves the modulation of several multifaceted complex pathways that regulate host immunity. These include targeted disruption of T-cell function through production of molecules that inhibit effector T-cells (1), suppression of T-cell cytotoxic death receptors (2), and induction of proapoptotic molecules targeted at tumor infiltrating lymphocytes (3, 4). Other methods of immune evasion include inhibition of B-cell and dendritic cell function (5) as well as secretion of the immunosuppressive mediators, such as transforming growth factor-{beta}, interleukin-10 (IL-10),1 and cyclooxygenase-2 (COX-2)-derived PGE2 (6, 7). Finally, malignant cells evade host immunity through induction of complement regulatory proteins (CRPs) that function to inhibit complement-mediated cell death (8-10).

Complement protects the host against microbial invasion through opsonization, and its activation triggers humoral and cellular inflammatory responses (11, 12). Normal cells anchor CRPs in the plasma membrane to defend host tissues from autologous complement attack. These include membrane cofactor protein (CD46), protectin (CD59), and decay-accelerating factor (also known as CD55). Stimulation of C3 convertase is the most important step in complement activation. Under physiologic and pathologic conditions, DAF decreases mistargeted complement attack by preventing C3 convertase from forming and triggering rapid inactivation of the enzyme (13).

Previous reports suggest that tumor cells induce CRPs as an effective mechanism to evade immune surveillance and complement-mediated cytotoxicity. Increased expression of CRPs has been described in several different malignancies, including colorectal (14), gastric (15), lung (16), renal (17), and breast (18) cancers. Specifically, increased DAF expression has been observed in 75% of colorectal cancers compared with matched normal tissue (19), and increased DAF expression has been associated with poor prognosis in patients with colorectal cancer (14). In addition to the angiogenic growth factors such as vascular endothelial growth factor and basic fibroblast growth factor (20), DAF expression is regulated by proinflammatory mediators such as lipopolysaccharide (21), tumor necrosis factor-{alpha} (22), and IL-1{beta} (23).

Prostaglandins modulate immune function through a variety of mechanisms (24, 25). PGE2 in particular has some known roles in the regulation of humoral and cellular immunity. PGE2 is generated from arachidonic acid by the enzymes COX-1 and COX-2. The inducible isoform, COX-2, is highly expressed at sites of inflammation (26, 27), and COX-2-derived PGE2 can signal via four distinct G-protein-coupled cell surface receptors (EP1-EP4) (28). Activation of EP2 and EP4 receptors leads to increased intracellular cyclic AMP (cAMP) levels through activation of G{alpha}s-coupled protein. High levels of cAMP stimulate the cAMP-dependent kinase, protein kinase A, which ultimately increases expression of cAMP-responsive genes. In contrast, the activation of EP3 leads to decreased cAMP levels, whereas EP1 does not directly regulate intracellular levels of cAMP but increases free cytosolic Ca2+ levels through activation of G{alpha}q.

Numerous reports have demonstrated increased expression of COX-2 in a variety of human malignancies (29, 30), and high COX-2 expression correlates with a poor clinical outcome (31). High levels of COX-2-derived PGE2 is associated with resistance to programmed cell death (32) as well as increased cell migration, proliferation, and angiogenesis (33). Immunosuppressive roles of PGE2 reported previously include suppression of T- and B-cell proliferation (34) as well as modulation of professional antigen-presenting cell activity (5). Finally, elevated PGE2 contributes to malignancy through immunosuppression of natural killer cell cytotoxicity (35, 36) and modulation of T-cell-derived cytokine production through inhibition of Th1 cytokines (interferon-{gamma} and IL-2) (37, 38) and stimulation of Th2 cytokines (IL-4, IL-5, and IL-10) (39).

We sought to identify PGE2-regulated genes downstream of elevated COX-2 activity in colon cancer. Using a variety of approaches, we present data in this study suggesting that decay-accelerating factor, a complement regulatory protein, is a direct target of PGE2 in LS174T colon carcinoma cells. This is the first indication that prostaglandins regulate components of the complement cascade, which may allow malignant cells to evade complement-mediated cytotoxicity and contribute to carcinogenesis. Ultimately, this novel observation may shed light on the adverse clinical outcomes of patients with high levels of tumor-derived cyclooxygenase-2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Prostaglandins (PGE2, PGA2, PGD2, PGF2{alpha}, thromboxane B2, and PGJ2) and antibody to COX-2 (catalogue number 160106) were obtained from Cayman Chemical (Ann Arbor, MI). LY294002, H-89, and cholera toxin were purchased from Calbiochem. Antibodies to DAF (catalogue number SC-9156) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal {beta}-actin antibody and 3,3-diaminobenzidine were obtained from Sigma.

Cell Culture—LS174T, HCT-15, and OVCAR-3 cells were purchased from the ATCC (Manassas, VA), and HCA-7 cells were a generous gift from Susan Kirkland. LS174T, HCT-15, and HCA-7 cells were maintained in McCoy's 5A medium containing 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a 5% CO2 atmosphere. OVCAR-3 cells were maintained in RPMI 1640 media containing 20% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a 5% CO2 atmosphere.

Northern Blotting—Total cellular RNA was isolated from cells by TRI reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer's protocol. Five micrograms of total RNA were fractionated with a MOPS-formaldehyde-agarose gel and transferred to Hybond N1 membrane (Amersham Biosciences). Following UV cross-linking, the blots were prehybridized for 30 min at 42 °C in Hybrisol I (Intergen Company, Purchase, NY), hybridized using 32P-labeled cDNA in the same buffer at 42 °C, and subjected to autoradiography. The 0.5-kb DAF (NM_000574 [GenBank] ) probe was amplified by reverse transcription-PCR using primers 5'-TTCAGGCAGCTCTGTCCAGT-3' (sense, 683-702) and 5'-TAAGTCAGCAAGCCCATGGT-3' (antisense, 1191-1210). The 0.4-kb actin (BC004251 [GenBank] ) probe was amplified by reverse transcription-PCR using primers 5'-TGGCACCACACCTTCTACAA-3' (sense, 317-336) and 5'-CATCTCTTGCTCGAAGTCCA-3' (antisense, 723-742).

Western Blotting—Cells were washed with PBS and lysed with radioimmune precipitation assay buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitors from Roche Diagnostics). Protein concentrations were measured using Bio-Rad reagent (Bio-Rad). Proteins were then separated on precast SDS-polyacrylamide gels and electrotransferred onto nitrocellulose membranes. Membranes were blocked in 5% milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) and incubated with primary antibody overnight at 4 °C. The membranes were then treated with horseradish peroxidase-conjugated secondary antibody and developed using an ECL kit (Amersham Biosciences).

Promoter Constructs, Transient Transfection, and Luciferase Assay—Various lengths of the human DAF promoter (40) spanning -724 to +80 bp with respect to the transcription initiation site were amplified using human genomic DNA (Promega, Madison, WI). For cloning purposes, the primer pairs containing BglII (forward) and HindIII (reverse) restriction sites at the 5' site were amplified by PCR, digested with BglII/HindIII enzymes, and cloned into pGL3-Basic firefly luciferase reporter vector (Promega). Substitution mutations at the cAMP response element (CRE) binding site were generated by PCR using (forward) 5'-TTTGTCCCACCCTTGGTGATTCAGAGCCCCAGCCCAGAC-3' and (reverse) 5'-GTCTGGGCTGGGGCTCTGAATCACCAAGGGTGGGACAAA-3' oligonucleotides. Following amplification of the -383/+80 promoter construct using Pfu turbo DNA polymerase (Stratagene), the PCR product was digested with DpnI enzyme and transformed into DH5{alpha}-competent cells. Small cultures from individual colonies were used for Miniprep DNA preparation (Qiagen). Each construct was sequence-verified before further evaluation. For luciferase assays, 1.3 x 105 LS174T cells were cultured in a 12-well plate 24 h before transfection. The transfection was carried out using Lipofectamine (Invitrogen) in serum-free media containing 100 ng of reporter plasmid and 5 ng of Renilla luciferase reporter plasmid pRLSV40 as an internal control following the manufacturer's protocol. This transfection mixture was added to cells, and the plates were incubated at 37 °C for 24 h. Prostaglandins and the other experimental reagents were added after 24 h and incubated for a further 24 h. Firefly and Renilla luciferase activities were measured using a dual luciferase assay kit (Promega) and a luminometer. Firefly luciferase values were normalized to Renilla values.

Immunohistochemistry—Six-week-old male ApcMin+/- mice were given 300 µg/kg PGE2 or sterile PBS vehicle control per os twice a day for 7 weeks. Pilot experiments consistently demonstrated that 300 µg/kg PGE2 achieved 2-3-fold higher levels of circulating and intestinal-derived PGE2 when compared with vehicle-treated control mice (data not shown). After 7 weeks, the mice were sacrificed by CO2 asphyxiation, and the entire intestine was dissected, washed in PBS, and immediately fixed in 10% neutral buffered formalin overnight at room temperature for paraffin embedding. Sections (5 µm) were dewaxed with xylene and rehydrated; the epitopes were revealed by microwave. Once endogenous peroxidase activity was quenched, nonspecific immunoglobulins were blocked with normal goat serum (Vector Laboratories, Burlingame, CA), and samples were incubated overnight at 4 °C with rabbit anti-human DAF primary antibody (1:50 dilution), which cross-reacts with mouse DAF protein. Negative controls received no primary antibody. The Vectastain ABC peroxidase system (Vector Laboratories) was used for immunodetection following the manufacturer's instructions, and immunolocalization was visualized with the peroxidase substrate 3,3-diaminobenzidine. Samples were counterstained with hematoxylin and mounted. All results were verified by a blind independent second observer.

Human Colorectal Tissue Samples—Human colorectal tumor specimens were obtained from surgical resections with Vanderbilt University internal review board approval. For each tumor sample, matched adjacent normal mucosa was collected for comparison. All samples were snap frozen and stored in liquid nitrogen until use. RNA preparation from tissues was performed using TRI reagent as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PGE2 Induces DAF mRNA and Protein in LS174T Cells—To evaluate the temporal profile of PGE2-mediated DAF expression, we conducted a time course following PGE2 treatment (1 µM). Northern blot analysis revealed that PGE2 rapidly induces DAF expression, beginning at 1.5 h (5-fold) and reaching a maximum by 4 h (20-fold) (Fig. 1A). PGE2 treatment increased DAF protein levels by 4 h (2-fold), and DAF expression remained elevated for 24 h (Fig. 1B). Furthermore, adding PGE2 induced DAF expression in a dose-dependent manner (Fig. 1C). mRNA levels were maximal at 2 µM (8-fold), whereas protein levels peaked at 10 µM (11-fold) PGE2 (Fig. 1D).



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  		FIG. 1.
PGE2 induces DAF in LS174T cells. LS174T cells were cultured in serum-free media for 48 h prior to PGE2 (1 µM) treatment. Total RNA was isolated following harvest of the cells at the indicated time points. Equal amounts of RNA were loaded, and the levels of DAF mRNA were determined by Northern blot analysis (A). Following the isolation of total cellular protein, equal amounts of protein were separated by SDS-PAGE and visualized with DAF antibody (B). LS174T cells were cultured in serum-free media for 48 h prior to PGE2 (0.05-10 µM) stimulation, and cells were harvested after 4 h. The levels of DAF mRNA were determined by Northern blot analysis as noted above (C). Total cellular proteins were separated by SDS-PAGE and visualized with DAF antibody (D).

 
PGE2 also increased human DAF promoter activity in LS174T cells. Based on the identified transcription initiation start site (41), a series of 5' DAF promoter deletion constructs were generated to evaluate the promoter activity of DAF in response to PGE2 treatment using reporter assays. The activity of these promoter constructs, which was assessed by their ability to drive luciferase expression, is shown in Fig. 2A. Transfection of the DAF -383/+80 construct resulted in maximal (~15-fold) PGE2-mediated luciferase activity compared with the other constructs. Significantly, deletion of the CRE dramatically reduced luciferase activity, suggesting that PGE2-dependent DAF induction is mediated by cAMP. This was further verified by substitution mutation of the CRE element from TGACACAG to TGATTCAG in the -383/+80 construct. This two-base mutation in the CRE decreased PGE2 induction of DAF by 90% relative to the wild-type construct (Fig. 2B). PGE2 also induced DAF promoter activity in a dose-dependent manner with maximal activity at a concentration of 0.5 µM (Fig. 2C).



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  		FIG. 2.
PGE2 mediates an increase in DAF promoter activity in LS174T cells. A, construction of the following five deletion mutants of the DAF promoter: -805/+80, -660/+80, -516/+80, -383/+80, and -205/+80. LS174T cells were cultured in 12-well plates and transiently cotransfected with a series of DAF promoter-luciferase reporter constructs and internal standard controls. Transfected cells were treated with PGE2 (1 µM) for 24 h prior to measurement of firefly luciferase activity normalized to Renilla luciferase activity, as described under "Experimental Procedures." B, CRE mutation in the DAF promoter. LS174T cells were transfected with a 2-base mutation of the CRE-like site in the DAF promoter construct (-383/+80) and treated with PGE2 (1 µM) for 24 h. Luciferase activity was measured as described above, and the experiments were repeated three times. C, LS174T cells were transfected with a -383/+80 construct and treated with increasing concentrations of PGE2. Luciferase activity was measured as described above, and the experiments were repeated three times.

 
PGE2 Stimulates DAF in LS174T Cells in a cAMP/Protein Kinase A-dependent Mechanism—Four G-protein-coupled receptors are known to mediate PGE2 signal transduction via distinct second-messenger pathways. To elucidate the specific mechanism by which PGE2 induces DAF expression, we utilized a compound known to elevate intracellular cAMP levels. Incubation with 1 µg/ml cholera toxin, which activates adenylate cyclase and increases cAMP levels, significantly induced DAF expression (25-fold) (Fig. 3A). Induction of DAF by PGE2 via cAMP signaling was further evaluated using H-89, a selective inhibitor of the cAMP-dependent protein kinase A. H-89 blocked the induction of DAF by PGE2 (Fig. 3). These findings are consistent with the hypothesis that PGE2 induces DAF expression in a cAMP/protein kinase A-dependent manner.



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  		FIG. 3.
PGE2 stimulates DAF in LS174T cells in a cAMP/protein kinase A-dependent mechanism. LS174T cells were cultured in serum-free media for 48 h prior to treatment with various reagents. Inhibitors (10 µM each H-89 and LY294002 (Ly)) were added 1 h before PGE2 stimulation. The levels of DAF mRNA were determined by Northern blot analysis (A). Equal amounts of protein were separated by SDS-PAGE and visualized with DAF antibody (B). LS174T cells were transfected with a -383/+80 construct and treated with and without protein kinase A inhibitor (H-89) prior to the addition of PGE2 (1 µM). Luciferase activity was measured as described above. The experiments were repeated three times, and representative results are shown (C). CTL, control.

 
We further evaluated PGE2 stimulation of DAF by other signaling pathways known to affect cAMP levels. We have shown previously that PGE2 has a positive effect on the phosphatidylinositol 3'-kinase (PI3K) and protein kinase B (AKT) pathways (42). Others have demonstrated that activation of the PI3K/AKT pathway reduces cAMP levels through increasing the activity of cyclic nucleotide phosphodiesterase 3B (43). Consistent with a model in which inhibiting the PI3K/AKT/phosphodiesterase 3B pathway increases cAMP levels (44), pretreatment with the PI3K inhibitor LY294002 prior to adding PGE2 induced a robust increase in DAF expression (40-fold) in LS174T cells (Fig. 3A).

PGD2 and PGJ2 Also Stimulate DAF Expression—The effect of PGE2 prompted us to assess the effect of other prostaglandins on the regulation of DAF expression. We treated LS174T cells with a variety of prostaglandins (1 µM) and measured DAF expression. Although PGA2, PGF2{alpha}, and thromboxane B2 had no effect, PGD2 (38-fold) and its metabolic product, PGJ2 (43-fold), stimulated DAF mRNA expression in a similar concentration as that of PGE2 (24-fold) (Fig. 4A). Protein levels of DAF also increased with PGD2 (1.8-fold) and PGJ2 (2.2-fold) (Fig. 4B). These data complement the profile of LS174T prostanoid receptor expression, which consists of receptors for PGE2 (EP2 and -4) and PGD2 (D prostanoid) (data not shown). The Northern and Western blot results were further corroborated with DAF promoter studies (Fig. 4C)



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  		FIG. 4.
A variety of prostaglandins stimulate DAF expression. LS174T cells were cultured in serum-free media for 48 h prior to treatment with various prostaglandins (1 µM). Cells were harvested after 4 h. Total RNA was isolated, and equal amounts of RNA were loaded. DAF mRNA levels were determined by Northern blot analysis (A). Following the isolation of total cellular protein, equal amounts of protein were separated by SDS-PAGE and visualized with DAF antibody (B). LS174T cells were transfected with a -383/+80 construct and treated with different prostaglandins (1 µM). Luciferase activity was measured after 24 h as described above. The experiments were repeated three times, and representative results are shown (C). TXB2, thromboxane B2.

 
DAF Is Induced by PGE2 in Other Cancer Cell Lines—To assess the ability of PGE2 to induce DAF in other cancer cells, we evaluated HCA-7 (colon), HCT-15 (colon), and OVCAR-3 (ovary) cells. This panel of cell lines was selected because of their differential production of PGE2 downstream from the two cyclooxygenase isoenzymes, COX-1 and COX-2. HCA-7 cells express high levels of COX-2 and endogenous PGE2 (4 ng/105 cells) (45), and concomitantly high levels of DAF are found in these cells as well (Fig. 5A). Consistent with this model, treatment with exogenous PGE2 (1 µM) did not further induce DAF mRNA levels (Fig. 5A). On the other hand, OVCAR-3 cells express high levels of COX-1, leading to a moderate amount of PGE2 production (0.25 ng/105 cells) (46). Consequently, adding exogenous PGE2 induced DAF expression over basal levels (2-fold). Finally, HCT-15 cells do not express either COX-1 or COX-2, producing low prostaglandins levels (47). Thus, adding PGE2 induced DAF expression (2-fold).



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  		FIG. 5.
DAF is expressed in multiple cancer cell lines and is inhibited in COX-2-expressing cells by an NSAID. HCA-7, OVCAR-3, and HCT-15 cells were cultured in serum-free media for 24 h prior to the addition of PGE2 (1 µM). Cells were harvested after 24 h. Total RNA was isolated, and equal amounts of RNA were loaded. DAF mRNA levels were determined by Northern blot analysis (A). HCA-7 cells were grown in 6-well plates for 24 h prior to the addition of serum-free media containing indomethacin (Indo) (10 µM) and grown for 72 h. Following the isolation of total cellular protein, equal amounts of protein were separated by SDS-PAGE and visualized with DAF antibody (B). CTL, control.

 
We could also modulate DAF expression by inhibiting COX activity with the non-selective NSAID indomethacin in HCA-7 cells. DAF protein levels were reduced following treatment with indomethacin (3-fold) (Fig. 5B), and PGE2 levels mirrored the decrease in DAF expression (data not shown). PGE2 levels were reduced by 5-fold following 24 h of treatment with indomethacin and remained undetectable from 48 to 72 h. The addition of exogenous PGE2 restored DAF expression, suggesting that PGE2 can rescue DAF expression upon COX inhibition. These data support the hypothesis that DAF induction is mediated by cyclooxygenase-derived PGE2.

In Vivo Studies of DAF Expression—ApcMin+/- mice are known to develop multiple intestinal polyps at ~15 weeks of age (48). Although COX-2 and PGE2 levels are relatively low initially, after 12 weeks of age these levels are elevated dramatically. We evaluated the correlation between COX-2 and DAF expression in intestinal polyps. Compared with adjacent normal tissue, expression of both COX-2 and DAF was greatly increased in the polyps (Fig. 6, A and B).



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  		FIG. 6.
PGE2 mediates DAF expression in vivo. Total RNA from polyps (P) and normal (N) adjacent tissue of two ApcMin+/- mice were isolated and examined by Northern blot analysis. The RNA was transferred to nitrocellulose filters, which were exposed to DAF and COX-2 probes (A). Following the isolation of total cellular protein from polyps and normal tissue from ApcMin+/- mice, equal amounts of protein were separated by SDS-PAGE, transferred to filters, and visualized with DAF and COX-2 antibody (B). Immunohistochemistry analysis of DAF immunoreactivity in small intestinal tissues of ApcMin+/- mice treated with (C, I and II) and without (C, III and IV) PGE2. Tissue sections were stained with rabbit anti-human DAF primary antibody and sections were counterstained with hematoxylin.

 
We next evaluated the ability of PGE2 to effect DAF expression as part of the polyp formation process. For these experiments, we treated 5-week-old ApcMin+/- mice with PGE2 for 7 weeks and then examined DAF expression by immunohistochemical analysis of intestinal sections. Compared with vehicle-treated control mice (Fig. 6C, III and IV), 300 µg/kg PGE2 treatment (Fig. 6C, I and II) induced DAF expression exclusively on the luminal surface of the small intestine. DAF expression was heterogeneous, revealing apical surface immunolocalization in differentiated villus epithelial cells. Sections from PBS-treated mice stained negatively for DAF, as did sections from PGE2-treated mice incubated without primary antibody as a negative control (data not shown).

Finally, assessment of DAF in human colon cancers and matched normal tissues revealed increased expression levels in malignant tissues. Northern blot analysis shows increased DAF expression in the large majority (14 of 16) of colon carcinoma samples as compared with adjacent normal mucosa (Fig. 7).



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  		FIG. 7.
DAF expression is induced in human colorectal tumors. Total RNA was isolated from 16 individual human colorectal cancer tissues and corresponding normal mucosa. Equal amounts of mRNA were analyzed for DAF expression. N, normal mucosa; T, tumor tissue.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Studies exploring the relationship between host immune status and susceptibility to cancer represents an important field with tremendous potential to improve human health (49). The signaling networks involved are complex, and the molecular mechanisms modulating inflammation, immune surveillance, and tumorigenesis remain poorly understood. The present study sought to examine the hypothesis that two established mediators of immunosuppression in cancer, prostaglandin E2 and decay-accelerating factor, are connected in a causal and directional relationship.

The inducible cyclooxygenase isoenzyme, COX-2, is significantly over-expressed at sites of inflammation and in various malignant tissues, with concomitant overproduction of the major arachidonate metabolite, PGE2. A large body of evidence has revealed a 40-50% reduction in colorectal cancer in individuals taking NSAIDs regularly. These effects are due, at least in part, to the inhibition of the cyclooxygenase enzymes and decreased production of PGE2. Although increased levels of COX-2-derived PGE2 are found in several different solid tumors, all of the effector genes downstream of this bioactive lipid are not well understood. We sought to carefully examine the role of COX-2 and prostaglandins in epithelial biology and carcinogenesis by identifying PGE2-regulated genes that mediate the effects of elevated COX-2 expression in colorectal cancer.

The well studied roles of COX-2 in malignant and metastatic disease have been shown to involve inhibition of apoptosis, stimulation of angiogenesis, and promotion of tumor invasion. However, the role of PGE2 in subversion of the immune system has been less well characterized. Using a variety of methods, we describe for the first time the ability of PGE2 to induce a major complement regulatory protein, DAF, through a cAMP/protein kinase A-dependent mechanism in human colon cancer cells. Polyps from ApcMin+/- mice showed increased DAF expression compared with adjacent normal tissue that correlated extremely well with COX-2 expression. In addition, ApcMin+/- mice treated with PGE2 for 7 weeks were also found to have increased DAF expression in the intestinal epithelium, and paired samples from patients with sporadic colorectal cancer showed increased DAF expression compared with matched normal mucosa.

DAF is known to play several important roles in modulating host immunity and tumorigenesis. All human cells express one or more surface molecules that regulate activation of C3, the main constituent of the complement cascade. In the rate-limiting step initiating the complement cascade, C3 convertase enzymatically cleaves C3 into two active components, C3a and C3b. As a complement regulatory protein, DAF regulates this important step through inhibition of C3 convertase and subsequent complement activation. While C3b serves as an opsonin facilitating phagocytosis of microbial invaders, C3a functions as a neutrophil-activating anaphylactic toxin (50). This activation, in turn, further amplifies the host inflammatory response through the generation of reactive oxygen species, cytokines, and arachidonate metabolites (51). Similarly, insertion of one complement-derived membrane attack complex subunit into tumor cell plasma membrane stimulates the synthesis of PGE2 and thromboxane B2 (52). These mutually reinforcing effects may further increase production of PGE2 within the tumor microenvironment, yielding a paracrine loop that mediates increased DAF expression on the surface of tumor cells. Significantly, DAF expression is often much greater on tumor cells than in surrounding normal tissue, particularly in colorectal cancers (19). In addition to providing complement resistance, high levels of DAF can facilitate the dissemination of metastasizing tumor cells once in circulation (8). Thus, several mechanisms may underlie the association of PGE2-mediated DAF induction with immunosuppression and tumorigenesis.

Conversely, loss of DAF expression has been linked to paroxysmal nocturnal hemoglobinuria, a condition characterized by complement-mediated hemolysis through autologous complement attack (53, 54). Hyperacute rejection in xenografts is mediated in part through reduced activity of complement regulatory proteins, including CD59, membrane cofactor protein, and DAF (55). Recent studies involving transgenic DAF expression in pigs prior to xenotransplantation to humans indicate that hyperacute rejection of the transplant can be partially reversed (56). Gene deletion studies with DAF demonstrated impaired regulation of complement activation involving antibody-mediated cytotoxicity as well as increased susceptibility to autoimmune glomerular basement membrane damage (57, 58). These studies underscore the importance of DAF in modulating the immune system in vivo.

Data presented here provide the first evidence suggesting that PGE2 can protect tumor cells from autologous complement attack. Recent studies have demonstrated similar immunomodulatory effects using other compounds that regulate colon cancer cell cytotoxicity. For example, the potent anti-tumor effects of sodium butyrate, previously attributed to induction of tumor cell apoptosis (59), have recently been shown to include increased complement sensitivity because of inhibition of DAF expression (60). Both selective and non-selective NSAIDs possess potent proapoptotic effects to which colon cancer cells are particularly sensitive (61). By extension, NSAIDs may facilitate restoration of complement-mediated tumor cytotoxicity. Selective complement targeting through inhibition of CRPs has also been proposed as adjunct therapy for patients with several different cancers (62). Cervical cancer cells develop increased sensitivity to monoclonal immunotherapy upon suppression of DAF expression (63). Future studies will explore the susceptibility of DAF-/- mice to cancer.

The novel findings presented in this study suggest that adjunct treatment of colorectal cancer with NSAIDs in combination with immunotherapy may increase the overall efficacy of colorectal cancer treatment. Because of increased potency and reduced side effects, immunotherapy continues to gain momentum for the treatment of a variety of human cancers (64). Future studies will yield greater insight into which effector genes mediate tumorigenicity downstream of COX-2-derived PGE2. Future work in this area will reveal the mechanisms involved in the complex progression from chronic inflammation and immunosuppression to overt tumor formation in the intestine.


   FOOTNOTES
 
* This work was supported in part by United States Public Health Services Grants RO-DK-62112 and P0-CA-77839. 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. Back

Hortense B. Ingram Professor of Molecular Oncology and the recipient of a National Institutes of Health MERIT award (R37-DK47297). To whom correspondence should be addressed: Vanderbilt-Ingram Cancer Center, 691 Preston Bldg., 2300 Pierce Ave., Nashville, TN 37232-6838. Tel.: 615-343-0527; Fax: 615-936-6865; E-mail: raymond.dubois{at}vanderbilt.edu.

1 The abbreviations used are: IL, interleukin; COX, cyclooxygenase; PG, prostaglandin; DAF, decay-accelerating factor; CRE, cAMP response element; MOPS, 4-morpholinepropanesulfonic acid; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3'-kinase; NSAID, nonsteroidal anti-inflammatory drugs; CRP, complement regulatory protein; EP, E prostanoid; Th, T (cell) helper. Back


   ACKNOWLEDGMENTS
 
We thank the T. J. Martell Foundation and the National Colorectal Cancer Research Alliance for generous support. We also thank Dr. S. K. Dey for valuable input throughout this study.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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