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. 2011;6(12):e29060.
doi: 10.1371/journal.pone.0029060. Epub 2011 Dec 15.

Regulation of cysteinyl leukotriene receptor 2 expression--a potential anti-tumor mechanism

Cecilia Magnusson  1 , Astrid M Bengtsson, Minghui Liu, Jian Liu, Yvonne Ceder, Roy Ehrnström, Anita Sjölander
Affiliations

Regulation of cysteinyl leukotriene receptor 2 expression--a potential anti-tumor mechanism

Cecilia Magnusson et al. PLoS One. 2011.

Abstract

Background: The cysteinyl leukotrienes receptors (CysLTRs) are implicated in many different pathological conditions, such as inflammation and cancer. We have previously shown that colon cancer patients with high CysLT(1)R and low CysLT(2)R expression demonstrate poor prognosis. Therefore, we wanted to investigate ways for the transcriptional regulation of CysLT(2)R, which still remains to be poorly understood.

Methodology/principal findings: We investigated the potential role of the anti-tumorigenic interferon α (IFN-α) and the mitogenic epidermal growth factor (EGF) on CysLT(2)R regulation using non-transformed intestinal epithelial cell lines and colon cancer cells to elucidate the effects on the CysLT(2)R expression and regulation. This was done using Western blot, qPCR, luciferase reporter assay and a colon cancer patient array. We found a binding site for the transcription factor IRF-7 in the putative promoter region of CysLT(2)R. This site was involved in the IFN-α induced activity of the CysLT(2)R luciferase reporter assay. In addition, IFN-α induced the activity of the differentiation marker alkaline phosphatase along with the expression of mucin-2, which protects the epithelial layer from damage. Interestingly, EGF suppressed both the expression and promoter activity of the CysLT(2)R. E-boxes present in the CysLT(2)R putative promoter region were involved in the suppressing effect. CysLT(2)R signaling was able to suppress cell migration that was induced by EGF signaling.

Conclusions/significance: The patient array showed that aggressive tumors generally expressed less IFN-α receptor and more EGFR. Interestingly, there was a negative correlation between CysLT(2)R and EGFR expression. Our data strengthens the idea that there is a protective role against tumor progression for CysLT(2)R and that it highlights new possibilities to regulate the CysLT(2)R.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. There is an IRF-7 binding site and four E-boxes present in the CysLT2R putative promoter region.
(A) Schematic visualization of the CysLT2R promoter. Construct I contains the wildtype promoter region (1012 bp) and constructs II–V are various deletion constructs. Representative Western blots from three different experiments with the intestinal epithelial cell line Int 407 and the colon cancer cell lines Caco-2 and SW 480 were analyzed as follows. (B) The blot shows the levels of IFNαR1, and A431 cells were used as a negative control while HCT116 cells were used as a positive control. (C) The blot shows the levels of EGFR protein expression, A431 cells were used as a positive control and MCF7 cells were used as a negative control, and (D) the blot shows the levels of CysLT2R protein expression.
Figure 2
Figure 2. IFN-α induces CysLT2R luciferase reporter activity whereas EGF suppresses it.
(A) CysLT2R luciferase activity following transient transfection with pGL-3-CysLT2R construct in Int 407 cells, with or without treatment with 500–2000 U/ml IFN-α for 24 h. (B) Int 407 cells transfected with wildtype CysLT2R promoter or CysLT2R promoter deletion constructs. Cells were grown in the presence of IFN-α (1000 U/ml) or in the presence of EGF (100 ng/ml) for 24 h (C). In brief, followed by stimulation with IFN-α or EGF for 24 h and measurement of luciferase activity (relative luciferase units). Results shown are the mean ± SD of at least three independent experiments performed in triplicate. (D) Analysis of IRF-7 and E-box DNA binding activities by EMSA on CysLT2R promoter region in Int 407 cells. Modulation of IRF-7 DNA binding in cells stimulated with IFN-α (1000 U/ml). For competition assays, a 1000-fold molar excess of unlabeled oligonucleotides (Lane 4) or 10 µg anti-IRF-7 antibody (Lane 5) was added before addition of probe to the binding reactions, (left panel). Effect on E-box DNA binding in cells stimulated with 100 ng/ml EGF for 24 h. A 1000-fold molar excess of unlabeled oligonucleotides (Lane 4), or 10 µg anti-Snail antibody was added before addition of probe to the binding reactions (right panel, lane 5). (E) Real-time PCR quantification of mRNA expression of CysLT2R and immunoblot analysis of CysLT2R protein following treatment with or without 1000 U/ml IFN-α. (F) Real-time PCR quantification of mRNA expression of CysLT2R and immunoblot analysis of CysLT2R protein with or without treatment with EGF (100 ng/ml). The results are shown as means ± SD of at between three to teen separate experiments; *, P<0.05; **, P<0.01; ***, P<0.001 for one-way ANOVA or two tailed Student's t-test.
Figure 3
Figure 3. EGF induced activation of EGFR and snail in Int 407 cells.
(A) Western blot of Int 407 cells showing phosphorylation of EGFR after EGF (100 ng/ml) treatment for 5 or 15 min. (B) Western blot of Int 407 cells showing phosphorylation of snail after EGF treatment (100 ng/ml) for 12–24 h. (C) Real-time PCR quantification of mRNA expression of CysLT1R and CysLT2R with and without EGF (100 ng/ml) treatment. The results are shown as means ± SD of at least three separate experiments; *, P<0.05; **, P<0.01 and ***, P<0.001.
Figure 4
Figure 4. CysLT2R signaling mediates anti-tumorigenic effects.
(A) An alkaline phosphatase activity assay was used to determine the differentiation of Caco-2 cells. Cells were treated with IFN-α (500–2000 U/ml) and/or LTC4 (40 nM) for 72 h. The alkaline phosphatase activity was determined by measuring the absorbance at 405 nm due to formation of p-nitrophenol. (B) QPCR quantification of mRNA expression of MUC2 with or without treatment with LTC4 (40 nM), IFN-α (1000 U/ml), pretreatment for 30 min with CysLT2R inhibitor AP-100984 (1 µM) or CysLT1R inhibitor Montelukast (1 µM), in Caco-2 cells. (C) Caco-2 cells were incubated with LTC4 (40 nM) and/or IFN-α (1000 U/ml) for 48 h in medium containing 1.5% serum. To determine proliferation by thymidine uptake, [methyl-3H] thymidine (0.5 µCi/well) was added to the wells during stimulation. (D) Cell migration was analyzed with Int 407 cells grown in the presence or absence of EGF (100 ng/ml) and/or LTC4 (40 nM). The cells were allowed to invade a collagen gel on top of a Boyden chamber for 18 hrs. The results are shown as means ± SD of at least three different experiments; *, P<0.05; **, P<0.01; ***, P<0.001.
Figure 5
Figure 5. Representative IFNα/βR1 and EGFR staining in normal human colon tissue and colorectal adenocarcinomas.
(A) Top row, shows the degree of IFNα/βR1 staining of carcinomas. Bottom row shows the degree of EGFR staining of carcinomas (+/− to +++, 10× objective). (B) Distribution of high (++, +++) and low (+/−, +) IFNα/βR1 staining intensities of tumors in Duke's A, B and C, and (C) of EGFR. Samples are assessed according to total IFNα/βR1 and EGFR staining. Statistics are based on tumors from 78 patients that were included in the array. (D) Representative pairs of control and tumor immunostaining from a Duke's C patient stained with IFNα/βR1, EGFR and CysLT2R.
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