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Cell, Tumor, and Stem Cell Biology |
Cancer Research UK Colorectal Tumour Biology Research Group, Department of Cellular and Molecular Medicine, Faculty of Medical and Veterinary Science, Bristol University, Bristol, United Kingdom
Requests for reprints: Christos Paraskeva, Cancer Research UK Colorectal Tumour Biology Research Group, Department of Cellular and Molecular Medicine, Faculty of Medical and Veterinary Science, Bristol University, BS8 1TD, Bristol, United Kingdom. Phone: 117-928-7894; Fax: 117-928-7896; E-mail: c.paraskeva{at}bris.ac.uk.
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Abstract |
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Introduction |
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Whereas the association of COX-2 and PGE2 expression with colorectal cancer is strong, less is known about the downstream mechanisms responsible for their transforming properties. PGE2 is known to selectively bind four E-prostanoid (EP) receptor subtypes, termed EP1-4. These receptors regulate a number of cell signaling pathways. For example, the EP1 receptor binds a G-protein shown to increase Ca2+ and IP3 levels; the EP2 and EP4 receptor subtypes are Gs protein–linked and can stimulate cyclic AMP (cAMP) increases, whereas the major EP3 receptor subtype is Gi linked and therefore inhibits cAMP up-regulation (7). The effects of PGE2 on cell growth are therefore dependent on its net second messenger response, which in-turn is depend upon ligand concentration, receptor-ligand affinity, as well as target cell EP receptor expression (8). Recently, studies have suggested roles for EP receptor–mediated PGE2 signaling in colorectal cancer. For example, studies in mice have suggested a role for each receptor subtype in intestinal tumor formation (9–13). Roles for individual EP receptor subtypes have also been reported in the invasion, migration, and growth of carcinoma cells from tumor types, including colorectal, endometrial, breast, and lung (8).
Signalling via the EP4 receptor is associated with intestinal neoplasia. PGE2 stimulates the proliferation and motility of LS174T colorectal carcinoma cells through the EP4-dependent activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling (14). Furthermore, ACF formation in EP4–/– mice following AOM treatment is reduced to 56% of the wild-type level (12). The same study also showed a similar reduction in AOM-induced polyp formation in mice wild type for EP4 receptor expression by the ad libitum administration of ONO-AE2-227 (an EP4 receptor antagonist), whereas plating efficiency studies showed 1 µmol/L ONO-AE1-329 (an EP4 receptor agonist) to enhance HCA/7 colony number in vitro by 1.8-fold. The link between the EP4 receptor and colorectal cancer is further strengthened by combinatorial antagonism of the EP1 and EP4 receptors in Apc1309 mice, resulting in a greater reduction in polyp size than EP1 antagonism alone (15).
Although there is evidence for the importance of the EP4 receptor in intestinal carcinogenesis, there have been no reports of whether the EP4 receptor is overexpressed in human colorectal cancers (12), nor whether levels of EP4 receptor influence markers of malignant potential, such as anchorage-independent growth or a tumor growth response to PGE2. COX-2 and subsequently PGE2 levels are increased during colorectal carcinogenesis. Although PGE2 can promote the growth of colorectal cancer cells, there have been no studies examining the growth response of human adenoma cell lines to PGE2 or whether the response changes during tumor progression. There are some contradictory reports of the effects of PGE2 on epithelial cell proliferation, which could be due to cell line specific differences in EP receptor expression (8). Here, we provide in vivo and in vitro evidence that human EP4 protein expression increases with the progression of normal colonic epithelium to carcinoma. We also report that increased EP4 receptor expression transforms an anchorage-dependent adenoma cell line to anchorage independence and switched it to being growth stimulated by levels of PGE2, which were inhibitory to the parent adenoma cell line. In light of recent reports of the increased risk of adverse cardiovascular events following long-term COX-2 inhibition (16), findings presented here highlight the EP4 receptor as a possible target for the selective inhibition of PGE2-driven colorectal tumor progression, which may have advantages over COX-2 inhibition in colorectal cancer prevention/therapy.
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Materials and Methods |
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Treatments and measurement of cell yield. Cells were treated with PGE2 and/or ONO-AE2-227 (an EP4 receptor antagonist; ref. 12). ONO-AE2-227 was used with the kind permission of ONO Pharmaceuticals (Osaka, Japan). PGE2 (Sigma, Poole, Dorset, United Kingdom) and ONO-AE2-227 were reconstituted as 10 mmol/L stock solutions in ethanol and stored in aliquots at –20°C. Final concentrations were prepared in standard treatment medium [DMEM F-12 NUT-MIX (Life Technologies, Paisley, UK), 2% fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine] together with an appropriate volume of ethanol to attain a concentration of 0.1% (v/v) ethanol across all treated and vehicle "control" flasks. Cells were treated in triplicate with either 10, 1, 0.5, and 0.1 µmol/L PGE2 alone; 100 nmol/L ONO-AE2-227 alone; or with 10, 1, 0.5, and 0.1 µmol/L PGE2 and 100 nmol/L ONO-AE2-227 in standard treatment medium for 72 hours. All experiments were done in triplicate and subjected to statistical analysis.
Statistical analysis. The response of adenomas and carcinomas to PGE2 and ONO-AE2-227 (either alone or in combination) was compared by a two-way ANOVA of cell yield versus concentration. For the comparison of anchorage-independent growth status between parent and EP4-transfected adenoma cell lines, a two-way ANOVA was also conducted (colony number versus cell line).
SDS-PAGE Western blotting. Western blotting was done using standard techniques as described previously (20). EP4 was detected using a rabbit polyclonal antibody at 1:500 (Alpha Diagnostic International, San Antonio, TX). A mouse monoclonal 
-tubulin antibody (Sigma) was used to assess gel loading.
Reverse transcription-PCR. RNA was extracted from 5 x 106 cells by RNeasy Mini Kit with on column DNase treatment (Qiagen GmbH, Hilden, Germany) according to the recommendations of the supplier. Single-stranded cDNA was synthesized for 1.5 hours at 37°C in a 50-µL reaction containing 1 µg RNA, 1 µg oligo-dT primer, 40 units Moloney murine leukemia virus reverse transcriptase, 80 units rRNasin (Promega Corp., Madison, WI), and 0.2 µmol/L each deoxynucleotide triphosphate. PCR primer pairs for each EP receptor subtype were designed as in Table 1 . PCR reactions (25 µL) contained 2 µL cDNA solution, 1x PCR Master Mix (Promega), and 100 nmol/L primers. PCR used a Genius apparatus (Techne, Inc., Princeton, NJ) for 35 cycles. As internal standard, RT-PCR was carried out with primers hybridizing to the ß-actin cytoskeletal protein. Control PCR was done directly on RNA without the step of cDNA synthesis; no amplified DNA fragment was detected in this case.
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Tissue sections (4 µm) were treated with rabbit polyclonal EP4 antibody (Alpha Diagnostic International) at 1:500 after incubation with 5% goat serum. This was followed by incubation in biotinylated goat anti-rabbit secondary antibody (1:250) for 30 minutes at room temperature before the addition of peroxidase-conjugated streptavidin. Products were visualized by the addition of the substrate diaminobenzidine. As a negative control, the primary antibody was omitted. Samples of inflamed mucosa from cases of ulcerative colitis were used as positive controls because these have previously been shown to express EP4 (21). Sections were also incubated with EP4 blocking peptide (Alpha Diagnostic International) to confirm specificity of staining (data not shown). Sections were graded as strongly positive (++), moderately positive (+), or negative to weakly positive (+/–). The slides were scored by two observers (M.M. and I.W.) independently. In the few cases where there was a discrepancy, these were reconsidered, and a consensus decision was reached.
Flow cytometry. Flow cytometry was used to quantify the cell surface expression of transfected EP4 receptor protein in RG/C2 adenoma cells. EP4 expression was detected using an EP4 rabbit polyclonal (Alpha Diagnostic International) and FITC-conjugated secondary antibody (Sigma). Analysis was done using CellQuest software (BD-Europe, Cowley, Oxford, United Kingdom). A FACS Vantage SE (Becton Dickinson) was used for cell sorting.
Cloning and transfecting human EP4. Human EP4 was PCR amplified using the following primer sequences: sense, 5'-GCCAGCCACTATCATGTC-3' and antisense, 5'-TAGCCCTTCTGAGCACAG-3' and T-A cloned into pGEM-T Easy (Promega) before subcloning into pcDNA3.1 (Invitrogen, Paisley, United Kingdom). RG/C2 and HEK/293 cells were transfected using 5 µL LF2000 (LipofectAMINE LF2000, Life Technologies) and 5 µg DNA following growth to confluence and according to the manufacturer's instructions. The selection of G418-resistant adenoma clones started 48 hours after transfection. Individual colonies were removed by scraping and grown in separate T25 flasks with standard growth medium containing 400 µg/mL G418.
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Results |
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EP4 receptor expression by reverse transcription-PCR in adenoma and carcinoma cell lines. For a preliminary screen of the EP receptor profile in a series of colorectal tumor cell lines, a set of novel primers for EP1-4 were designed to amplify fragments of each receptor by reverse transcription-PCR (RT-PCR; each according to Genbank annotations nm-000955, nm-000956, nm-000957, and nm-000958 for EP1-4, respectively). To determine their specificity, primers were used to amplify cDNA isolated from the adenoma-derived cell line PC/AA/C1. Each of the four bands in Fig. 2A (EP1-4) correspond with the expected PCR fragment sizes. Primer specificity was confirmed by cloning and sequencing each band (data not shown). EP receptor mRNA expression was characterized in tumor cell lines as well as two in vitro models of colorectal tumor progression (Fig. 2B). The colonic adenoma-derived cell lines, which are anchorage dependent, nontumorigenic in athymic mice and responsive to transforming growth factor-ß (TGF-ß; refs. 17, 22), were screened for EP1-4 receptor expression. Three of three expressed EP1, three of three expressed EP2, one of three expressed EP3, and three of three expressed EP4 mRNA. Five colonic carcinoma derived cell lines (all tumorigenic in athymic mice) were also analyzed. All carcinomas expressed EP2 and EP4 transcript. EP3 was observed in four of five carcinomas, and the frequency of EP1 expression was one in five.
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19% and 35%, respectively, over control levels (Fig. 5A and B
). However, 10 µmol/L PGE2 had opposing effects on RG/C2 and HT29 cell yields, with HT29 carcinoma cell numbers being 45% greater than control levels after 3 days, whereas RG/C2 adenoma cell numbers are reduced by 15%. Interestingly, this trend was also observed in the PC/AA in vitro tumor progression model, where 0.5 µmol/L PGE2 stimulated the growth of both parental PC/AA/C1 adenoma (anchorage dependent and nontumorigenic) and its transformed derivative, PC/AA/C1/SB/10C carcinoma (anchorage independent and tumorigenic) cells by 25% and 40%, respectively. However, at the higher dose of 10 µmol/L, PGE2 reduced PC/AA/C1 adenoma cell yield by 20% while significantly increasing PC/AA/C1/SB/10C carcinoma cell yield by 55% (Fig. 5C). Thus, carcinomas are stimulated by the higher dose of 10 µmol/L PGE2, whereas both adenomas are inhibited and the transformation of an adenoma (PC/AA/C1) to a carcinoma (PC/AA/C1/SB/10C) cell line is accompanied by a change from growth inhibition to growth stimulation by 10 µmol/L PGE2. These results are of interest because as well as showing that PGE2 can promote the growth of adenoma as well as carcinoma cells, they also suggest that the response is determined by the malignant potential of the tumor and by the levels of PGE2. This could have important implications for clonal evolution and selection as discussed below.
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55% of the untreated control levels over 3 days (Fig. 5D). Interestingly, the addition of exogenous PGE2 dose dependently rescued HT29 carcinoma cells from ONO-AE2-227–induced growth inhibition at concentrations up to 1 µmol/L, at which concentration ONO-AE2-227 treatment resulted in no significant reduction in cell yield. However, HT29 cells antagonized by ONO-AE2-227 were growth inhibited by the higher dose of PGE2 (10 µmol/L) rather than being growth stimulated (Fig. 5D). Thus, when the EP4 receptor is antagonized with ONO-AE2-227 in HT29 colon cancer cells, they behave more like an adenoma than a carcinoma in response to the higher dose of PGE2. These data with the EP4 antagonist and the observation that adenomas are growth inhibited by the higher levels of PGE2, as noted above, suggest the response of the adenomas to the higher levels of PGE2 maybe limited by the relatively low expression of the EP4 receptor. Therefore, RG/C2 adenoma cells, which had been stably transfected with EP4, were treated with 0.1 to 10 µmol/L PGE2 to determine whether enforced expression of EP4 had altered their response to the higher dose of PGE2. Interestingly, RG/C2 adenoma cells with enhanced EP4 expression displayed a dose-dependent increase in cell yield up to and including 10 µmol/L PGE2, where a maximal increase in cell yield of 44% was observed (Fig. 5E), whereas no increases in cell yield were observed when vector-transfected adenoma cells were treated with 10 µmol/L (Fig. 5F). Taken together, these data suggest an important role for the EP4 receptor in mediating the growth response of colorectal tumor cells to PGE2 and that increased EP4 receptor expression maybe important for the proliferative response of colorectal tumors to relatively high levels of PGE2. Transfection of the EP4 receptor in the RG/C2 adenoma cell line transforms it into an anchorage-independent phenotype. The ability to grow anchorage independently in soft agar is an important characteristic of tumor progression and is typically associated with carcinoma but not adenoma cells (17). In this study, we have shown that the anchorage-dependent adenoma cell lines express lower levels of the EP4 receptor than the anchorage-independent carcinoma cells. Experiments were therefore done to determine whether enforced expression of the EP4 receptor could transform RG/C2 adenoma cells from an anchorage-dependent to anchorage-independent phenotype, hence promoting tumor progression.
Two clonogenic extremes of the RG series (nontransfected) were included as controls, RG/C2 parent adenoma cells, and RG/GV transformed adenoma cells. The results in Fig. 6 illustrate that the parent RG/C2 adenoma cells, as expected were anchorage dependent in the presence of exogenous PGE2, whereas the RG/GV cells as expected were anchorage independent. Interestingly, all three EP4 transfectants, RG/C2/EP4 clones 5 and 7 and the FACS sorted RG/C2/EP4/Clone 7F, similar to positive control RG/GV, displayed anchorage-independent growth, whereas two vector controls, RG/C2/pcDNA3.1/Clones 3 and 9 remained anchorage dependent (Fig. 6). Results shown represent cells cultured in agar for 3 weeks with 0.5 µmol/L PGE2, but similar results were achieved in the absence of exogenous PGE2 (data not shown). Thus, enforced expression of the EP4 receptor through transfection transformed the RG/C2 adenoma cell line to anchorage independence, a phenotype characteristic of carcinoma cell lines. Thus, increased expression of the EP4 receptor may be important in the acquisition of anchorage independence and hence promote tumor progression in colorectal carcinogenesis.
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Discussion |
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The high expression of EP4 protein in 100% of colorectal carcinomas and a subset of adenomas compared with normal colonic tissue reported in this study suggests that increased EP4 expression is important in human colorectal carcinogenesis. It has previously been reported that EP4-mediated PGE2 signals may also have distinct roles in murine colonic polyps, showing a 2.4-fold mRNA increase when compared with normal colonic epithelium by in situ hybridization (10). It also supports in vivo studies reporting increased lamina propria EP4 receptor expression, as well as a general increase in epithelial cell EP2 and EP3 expression in the colonic mucosa of individuals with ulcerative colitis (a disease that carries an increased risk of colorectal cancer and is also associated with increased PGE2 production; ref. 21). Similarly, in tissue sections from individuals with FAP, levels of expression of EP3 and EP4 receptor RNA in crypt epithelial cells of the large intestine are reported to be 2.8 times that of adjacent normal tissue (23).
In agreement with our in vivo findings, our in vitro data also showed that EP4 receptor expression was higher in anchorage-independent carcinoma cell lines than anchorage-dependent adenoma cell lines, and that EP4 expression increased in two in vitro models of the adenoma carcinoma sequence. This in vitro data provided a model in which the consequence of increased EP4 expression could be investigated. Interestingly, our results indicated that increased EP4 receptor expression is important for the acquisition of the anchorage-independent phenotype. When RG/C2, an adenoma cell line, was stably transfected with EP4, it became capable of anchorage-independent growth. This is of particular interest because anchorage independence is a phenotype associated with colorectal cancers and is a measure of autonomous cell growth, an important hallmark of cancer (27).
The majority of colorectal cancers and a subset of adenomas overexpress COX-2 (28), with its tumor-promoting properties believed, at least in part, to be mediated through increased PGE2. PGE2 has been shown to stimulate the growth of colorectal cancers, but although COX-2 and PGE2 levels are increased in some adenomas, the effect of PGE2 on the growth of human adenoma cell lines has not been reported. Studies presented here indicate that colorectal adenoma as well as carcinoma cell lines can be growth stimulated by the lower concentrations of PGE2 (up to 0.5 µmol/L). The positive effect of PGE2 on colorectal carcinoma cell growth is consistent with a number of studies that show dose-dependent proliferative effects of PGE2 on colorectal carcinoma cell lines, including HT29 (14, 29, 30) and HCA7, where it was shown that PGE2 increased HCA7 cell number in a dose-dependent manner up to and including 10 µmol/L (31). Although no studies of PGE2-mediated effects in vitro on human adenoma cell growth have previously been reported, our results using 0.1 to 0.5 µmol/L PGE2 are in agreement with in vivo studies, indicating PGE2 to be capable of increasing intestinal adenoma growth in ApcMIN mice (32).
Our observation that the adenoma cell lines, AA/C1 and RG/C2, are both inhibited by the higher doses of PGE2, whereas HT29 carcinoma, PC/AA/C1/SB10C (the transformed tumorigenic derivative of the AA/C1 adenoma cell line) and EP4 transfected RG/C2 cells are all growth stimulated, is of potential interest. This suggests that any growth advantage resulting from the over expression of COX-2 and PGE2 could at some stage during carcinogenesis become inhibitory to tumor cells. It is possible that unless increased levels of PGE2, brought about by increased COX-2, are at some point accompanied by an increased in EP4 receptor expression, paradoxically, the increased levels of PGE2 could switch from being tumor promoting to growth inhibitory. This hypothesis is supported by the fact that not only do carcinoma cell lines (which are stimulated by high levels of PGE2) have higher EP4 receptor levels, but that expression of EP4 receptor (by transfection) in the RG/C2 adenoma cell line results in the EP4-transfected cell line being stimulated by the higher levels of PGE2. Furthermore, when HT29 are treated with the EP4 receptor antagonist ONO-AE2-227, they are growth inhibited by 10 µmol/L PGE2 rather than stimulated, behaving more like an adenoma in their response to the higher levels of PGE2. This raises the interesting possibility that the response to PGE2, perhaps analogous to the TGF-ß system, changes during the adenoma carcinoma sequence. We have previously shown for example that adenoma cells alter their response from being inhibited by TGF-ß to resistant and sometimes even stimulated by TGF-ß during progression from adenoma to carcinoma (22). Gradients of very high levels of PGE2 (from both epithelial cells and stromal cells) in the microenvironment of a tumor maybe an important selective pressure during the adenoma carcinoma sequence and increased expression of EP4 receptor important for the tumor cells to switch from growth inhibition to growth promotion by relatively high levels of PGE2. Wang et al. have reported that PGE2 can amplify the expression of COX-2, a key enzyme in the PG biosynthetic pathway, in colorectal carcinoma cells through a positive feedback loop. This self-amplifying loop may explain why COX-2 is constitutively over expressed in the majority of colorectal cancers (31) and could contribute to gradients of high levels of PGE2 proposed above. It is interesting to note that a "bell-shaped" dose-response relationship for SW1116 colorectal cancer cells to PGE2 has previously been reported (29), suggesting differential activation of EP receptors at different PGE2 concentrations (8).
In summary, both in vivo and in vitro data presented in this article indicate an important role for the EP4 receptor in colorectal tumorigenesis. As PGE2 is often overproduced in colorectal tumors, increased EP4 receptor expression could represent an important step in the clonal evolution of colonic epithelial cells during the adenoma carcinoma sequence and contribute to the malignant phenotype. This highlights the EP4 receptor as a possible target for the selective inhibition of PGE2-driven colorectal carcinogenesis and, in the light of the recent side effects associated with the use of COX-2 inhibitors, represents an alternative target for the prevention and treatment of colorectal cancer.
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Acknowledgments |
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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.
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Footnotes |
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1 C. Paraskeva and A. Hague, unpublished data. 
Received 10/13/05. Revised 12/15/05. Accepted 1/10/06.
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) and its transformed carcinoma derivative PC/AA/C1/SB/10C (
). All cell lines were treated in triplicate with 0.1, 0.5, 1, and 10 µmol/L PGE2 for 72 hours. D, HT29 carcinoma cells treated with 0.1, 0.5, 1, and 10 µmol/L PGE2 for 72 hours in the presence of 100 nmol/L ONO-AE2-227 (EP4 antagonist). E, RG/C2 cells transfected with EP4 (RG/C2/EP4/Clone 7F) and (F) vector-transfected control (RG/C2/pcDNA3.1/Clone 9) treated in triplicate with 0.1, 0.5, 1, and 10 µmol/L PGE2 for 72 hours. In all cases, controls contained 0.1% (v/v) ethanol. Attached cell yields were represented as a percentage of the control. Columns, mean of three sets of triplicate flasks; bars, SD. Data were analyzed by a two-way ANOVA followed by Tukey's post hoc test, ***, P < 0.001, highly significant variation from control levels; *, P < 0.05, significant variation from control levels. Control levels are given as 100% or indicated by a dashed line.