Tumour suppressor 15-hydroxyprostaglandin dehydrogenase induces differentiation in colon cancer via GLI1 inhibition.

Inflammation is an established risk factor for colorectal cancer. We and others have shown that colorectal cancer patients with elevated cysteinyl leukotriene receptor 2 (CysLT2R) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) levels exhibit good prognoses. However, both CysLT2R and 15-PGDH, which act as tumour suppressors, are often suppressed in colorectal cancer. We previously reported that leukotriene C4 (LTC4)-induced differentiation in colon cancer via CysLT2R signalling. Here, we investigated the involvement of Hedgehog (Hh)-GLI1 signalling, which is often hyperactivated in colorectal cancer. We found that the majority of colorectal cancer patients had high-GLI1 expression, which was negatively correlated with CysLT2R, 15-PGDH, and Mucin-2 and overall survival compared with the low-GLI1 group. LTC4-induced 15-PGDH downregulated both the mRNA and protein expression of GLI1 in a protein kinase A (PKA)-dependent manner. Interestingly, the LTC4-induced increase in differentiation markers and reduction in Wnt targets remained unaltered in GLI1-knockdown cells. The restoration of GLI1 in 15-PGDH-knockdown cells did not ameliorate the LTC4-induced effects, indicating the importance of both 15-PGDH and GLI1. LTC4-mediated reduction in the DCLK1 and LGR5 stemness markers in colonospheres was abolished in cells lacking 15-PGDH or GLI1. Both DCLK1 and LGR5 were highly increased in tumour tissue compared with the matched controls. Reduced Mucin-2 levels were observed both in zebrafish xenografts with GLI1-knockdown cells and in the cysltr2-/- colitis-associated colon cancer (CAC) mouse model. Furthermore, GLI1 expression was positively correlated with stemness and negatively correlated with differentiation in CRC patients when comparing tumour and mucosal tissues. In conclusion, restoring 15-PGDH expression via CysLT2R activation might benefit colorectal cancer patients.

The WNT5A Agonist Foxy5 Reduces the Number of Colonic Cancer Stem Cells in a Xenograft Mouse Model of Human Colonic Cancer.

BACKGROUND: The wingless-type mammary tumour virus integration site 5A (WNT5A) agonist Foxy5 was shown in vitro to affect intracellular signalling implicated in the regulation of colonic cancer stem cells (CSCs). MATERIALS AND METHODS: In order to study whether Foxy5 can modulate CSCs, either HT-29 or Caco-2 human colonic cancer cells, both lacking endogenous WNT5A expression, were inoculated subcutaneously into nude mice. RESULTS: Foxy5 reduced the expression of the stem-cell marker aldehyde dehydrogenase and, interestingly, the specific colon CSC marker double cortin-like kinase 1. Foxy5 also reduced active ß-catenin and the expression of its downstream target Achaete Scute complex homolog 2, a CSC-preserving transcription factor. Foxy5 also reduced cyclo-oxygenase 2 expression, responsible for the formation of the CSC-promoting prostaglandin E2 (PGE2), but increased that of 15-hydroxyprostaglandin dehydrogenase expression, a PGE2-degrading enzyme. Accordingly, Foxy5 impairs both ß-catenin and PGE2 signalling, both of which have been implicated in promoting the niche of colonic CSCs. CONCLUSION: Our data suggest that Foxy5 can complement the traditional adjuvant chemotherapeutic treatment to which CSCs are resistant.

Cysteinyl leukotriene receptor 1 facilitates tumorigenesis in a mouse model of colitis-associated colon cancer.

Cysteinyl leukotriene receptor 1 (CysLT1R) has been shown to be up-regulated in the adenocarcinomas of colorectal cancer patients, which is associated with a poor prognosis. In a spontaneous model of colon cancer, CysLT1R disruption was associated with a reduced tumor burden in double-mutant female mice (ApcMin/+/Cysltr1-/-) compared to ApcMin/+ littermates. In the current study, we utilized a genetic approach to investigate the effect of CysLT1R in the induced azoxymethane/dextran sulfate sodium (AOM/DSS) model of colitis-associated colon cancer. We found that AOM/DSS female mice with a global disruption of the Cysltr1 gene (Cysltr1-/-) had a higher relative body weight, a more normal weight/length colon ratio and smaller-sized colonic polyps compared to AOM/DSS wild-type counterparts. The Cysltr1-/- colonic polyps exhibited low-grade dysplasia, while wild-type polyps had an adenoma-like phenotype. The Cysltr1-/- colonic polyps exhibited significant decreases in nuclear ß-catenin and COX-2 protein expression, while the normal crypts surrounding the polyps exhibited increased Mucin 2 expression. Furthermore, Cysltr1-/- mice exhibited an overall reduction in inflammation, with a significant decrease in proinflammatory cytokines, polyp 5-LOX expression and infiltration of CD45 leukocytes and F4/80 macrophages. In conclusion, the present genetic approach in an AOM/DSS model further supports an important role for CysLT1R in colon tumorigenesis.

The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model.

BACKGROUND: Colorectal cancer is one of the most common types of cancers worldwide. Recent studies have identified cancer-initiating cells (CICs) as a subgroup of replication-competent cells in the development of colorectal cancer. Although it is understood that an inflammation-rich tumor microenvironment presumably supports CIC functions, the contributory factors are not very well defined. The present study advances our understanding of the role of the eicosanoids leukotriene D4 (LTD4) and prostaglandin E2 (PGE2) in the tumorigenic ability of CICs and investigates the consequential changes occurring in the tumor environment that might support tumor growth. METHODS: In this study we used human HCT-116 colon cancer ALDH(+) cells in a nude mouse xenograft model. Protein expression and immune cell was determined in tumor-dispersed cells by flow cytometry and in tumor sections by immunohistochemistry. mRNA expressions were quantified using RT-q-PCR and plasma cytokine levels by Multiplex ELISA. RESULTS: We observed that LTD4 and PGE2 treatment augmented CIC-induced tumor growth. LTD4-and PGE2-treated xenograft tumors revealed a robust increase in ALDH and Dclk1 protein expression, coupled with activated ß-catenin signaling and COX-2 up-regulation. Furthermore, LTD4 or PGE2 accentuated the accumulation of CD45 expressing cells within xenograft tumors. Further analysis revealed that these infiltrating immune cells consisted of neutrophils (LY6G) and M2 type macrophages (CD206(+)). In addition, LTD4 and PGE2 treatment significantly elevated the plasma levels of cysteinyl leukotrienes and PGE2, as well as levels of IL-1ß, IL-2, IL-6, TNF-α and CXCL1/KC/GRO. In addition, increased mRNA expression of IL-1ß, IL-6 and IL-10 were detected in tumors from mice that had been treated with LTD4 or PGE2. CONCLUSION: Our data suggest that both LTD4 and PGE2 promote CICs in initiating tumor growth by allowing modifications in the tumor environment. Our data indicate that new therapeutic strategies targeting eicosanoids, specifically LTD4 and PGE2, could be tested for better therapeutic management of colon cancer.

High tumor mast cell density is associated with longer survival of colon cancer patients.

BACKGROUND: Inflammatory cells and inflammatory mediators play an important role in colorectal cancer (CRC). Previous studies have shown that CRC patients with increased expression of cysteinyl leukotriene receptor 1 (CysLTR1) have a poorer prognosis, and Cysltr1-/- mice display fewer intestinal polyps. However, the role of mast cells (MCs) in colon cancer progression remains unclear. The aim of the present study was to explore the relevance of MCs in CRC. MATERIAL AND METHODS: A tissue microarray from 72 CRC patients was stained with MC anti-tryptase and -chymase antibodies. Mouse colon tissue was stained with MC anti-tryptase antibody. Immunohistochemistry was used to identify MCs in patients and mice. RESULTS: Patient colon cancer tissue had in comparison with normal colon tissue a reduced number of MCs, predominantly of chymase-positive cells. Further analysis revealed that patients with a relative high MCD in their cancer tissues showed significantly longer overall survival compared to those with a low MCD [hazard ratio (HR) 0.539; 95% confidence interval (CI), 0.302-0.961]. Similar results were observed in subgroups of patients with either no distant metastasis (p = 0.004), or <75 years (p = 0.015) at time of diagnosis. Multivariate Cox analysis showed that MCD independently correlated with reduced risk of death in colon cancer patients (HR 0.380; 95% CI 0.202-0.713). Additionally, a negative correlation was found between cytoplasmic CysLTR1 expression and number of MCs. In agreement, in the CAC mouse model, Cysltr1-/- mice showed significantly higher MCs in their polyp/tumor areas compared with wild-type mice. CONCLUSION: A high MCD in cancer tissue correlated with longer patient survival independently from other risk factors for CRC. The concept that MCs have an anti-tumor effect in CRC is further supported by the findings of a negative correlation with CysLTR1 expression in patients and a high MCD in colon polyps/tumors from CysLTR1-/- mice.

Cysteinyl leukotriene 1 receptor influences intestinal polyp incidence in a gender-specific manner in the ApcMin/+ mouse model.

There is emerging literature emphasizing the role of inflammatory eicosanoids, including prostaglandins and leukotrienes, in cancer development. Increased expression of both the cysteinyl leukotriene receptor 1 (CysLTR1) and the enzyme responsible for the production of leukotrienes, 5-lipoxygenase, is associated with poor prognosis in patients with colorectal adenocarcinomas. Apc mutation is an early event in the development of sporadic and hereditary (familial adenomatous polyposis) colorectal cancer. We utilized the Apc(Min/+) mouse model of familial adenomatous polyposis/sporadic colorectal cancer to investigate the role of CysLTR1 in intestinal tumorigenesis by crossing Apc(Min/+) mice with mice lacking the Cysltr1 gene. We could observe a reduced tumor burden in the small intestine of double-mutant female (Cysltr1 (-/-) Apc (Min/+) ) but not double-mutant male mice, compared with gender-matched single-mutant (Cysltr1 (+/+) Apc (Min/+) ) mice. This reduction was in a Cysltr1-dependent manner, female double-mutant mice having significantly reduced tumor formation compared with control littermates. The female double-mutant phenotype was accompanied with decreased systemic inflammation, as evidenced by significantly reduced serum levels of prostaglandin E2 and CysLTs, as well as increased CD3(+)CD8(+) T-cell tumor infiltration. Furthermore, the reduced formation of polyps in double-mutant (Cysltr1 (-/-) Apc (Min/+) ) female mice could in part be explained by the cytotoxic action of CD3(+)CD8(+) T cells in the polyp and reduced nuclear accumulation of ß-catenin in the epithelium of small intestinal polyps. Our results stress the important role that CysLTR1 plays in colorectal cancer and its potential as a therapeutic target in cancer therapy.

Phenotype-based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis.

Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge Diverset(TM)library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4-10 µmin zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT1-2) at micromolar IC50values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.