Grace O'Callaghan

Prostaglandin E2 and the EP receptors in malignancy: possible therapeutic targets?

Elevated expression of COX‐2 and increased levels of PGE2 are found in numerous cancers and are associated with tumour development and progression. Although epidemiological, clinical and preclinical studies have shown that the inhibition of PGE2 synthesis through the use of either non‐steroidal anti‐inflammatory drugs (NSAIDs) or specific COX‐2 inhibitors (COXibs) has the potential to prevent and treat malignant disease, toxicities due to inhibition of COX‐2 have limited their use. Thus, there is an urgent need for the development of strategies whereby COX‐2 activity may be reduced without inducing any side effects. The biological effects of PGE2 are mediated by signalling through four distinct E‐type prostanoid (EP) receptors – EP1, EP2, EP3 and EP4. In recent years, extensive effort has gone into elucidating the function of PGE2 and the EP receptors in health and disease, with the goal of creating selective inhibitors as a means of therapy. In this review, we focus on PGE2, and in particular on the role of the individual EP receptors and their signalling pathways in neoplastic disease. As knowledge concerning the role of the EP receptors in cancer grows, so does the potential for exploiting the EP receptors as therapeutic targets for the treatment of cancer and metastatic disease.

Prostaglandin E2 stimulates Fas ligand expression via the EP1 receptor in colon cancer cells

Fas ligand (FasL/CD95L) is a member of the tumour necrosis factor superfamily that triggers apoptosis following crosslinking of the Fas receptor. Despite studies strongly implicating tumour-expressed FasL as a major inhibitor of the anti-tumour immune response, little is known about the mechanisms that regulate FasL expression in tumours. In this study, we show that the cyclooxygenase (COX) signalling pathway, and in particular prostaglandin E2 (PGE2), plays a role in the upregulation of FasL expression in colon cancer. Suppression of either COX-2 or COX-1 by RNA interference in HCA-7 and HT29 colon tumour cells reduced FasL expression at both the mRNA and protein level. Conversely, stimulation with PGE2 increased FasL expression and these cells showed increased cytotoxicity against Fas-sensitive Jurkat T cells. Prostaglandin E2-induced FasL expression was mediated by signalling via the EP1 receptor. Moreover, immunohistochemical analysis using serial sections of human colon adenocarcinomas revealed a strong positive correlation between COX-2 and FasL (r=0.722; P<0.0001) expression, and between EP1 receptor and FasL (r=0.740; P<0.0001) expression, in the tumour cells. Thus, these findings indicate that PGE2 positively regulates FasL expression in colon tumour cells, adding another pro-neoplastic activity to PGE2.

Targeting the EP1 receptor reduces Fas ligand expression and increases the antitumor immune response in an in vivo model of colon cancer

Despite studies demonstrating that inhibition of cyclooxygenase‐2 (COX‐2)‐derived prostaglandin E2 (PGE2) has significant chemotherapeutic benefits in vitro and in vivo, inhibition of COX enzymes is associated with serious gastrointestinal and cardiovascular side effects, limiting the clinical utility of these drugs. PGE2 signals through four different receptors (EP1–EP4) and targeting individual receptor(s) may avoid these side effects, while retaining significant anticancer benefits. Here, we show that targeted inhibition of the EP1 receptor in the tumor cells and the tumor microenvironment resulted in the significant inhibition of tumor growth in vivo. Both dietary administration and direct injection of the EP1 receptor‐specific antagonist, ONO‐8713, effectively reduced the growth of established CT26 tumors in BALB/c mice, with suppression of the EP1 receptor in the tumor cells alone less effective in reducing tumor growth. This antitumor effect was associated with reduced Fas ligand expression and attenuated tumor‐induced immune suppression. In particular, tumor infiltration by CD4+CD25+Foxp3+ regulatory T cells was decreased, whereas the cytotoxic activity of isolated splenocytes against CT26 cells was increased. F4/80+ macrophage infiltration was also decreased; however, there was no change in macrophage phenotype. These findings suggest that the EP1 receptor represents a potential target for the treatment of colon cancer.

CORK study in CF Sustained improvements in ultra-low dose chest CT scores post CFTR modulation with ivacaftor

Background Ivacaftor produces significant clinical benefit in patients with cystic fibrosis (CF) with the G551D mutation. Prevalence of this mutation at the Cork CF Centre is 23%. This study assessed the impact of cystic fibrosis transmembrane conductance regulator modulation on multiple modalities of patient assessment. Methods Thirty‐three patients with the G551D mutation were assessed at baseline and prospectively every 3 months for 1 year after initiation of ivacaftor. Change in ultra‐low‐dose chest CT scans, blood inflammatory mediators, and the sputum microbiome were assessed. Results Significant improvements in FEV1, BMI, and sweat chloride levels were observed post‐ivacaftor treatment. Improvement in ultra‐low‐dose CT imaging scores were observed after treatment, with significant mean reductions in total Bhalla score (P < .01), peribronchial thickening (P = .035), and extent of mucous plugging (P < .001). Reductions in circulating inflammatory markers, including interleukin (IL)‐1&bgr;, IL‐6, and IL‐8 were demonstrated. There was a 30% reduction in the relative abundance of Pseudomonas species and an increase in the relative abundance of bacteria associated with more stable community structures. Posttreatment community richness increased significantly (P = .03). Conclusions Early and sustained improvements on ultra‐low‐dose CT scores suggest it may be a useful method of evaluating treatment response. It paralleled improvement in symptoms, circulating inflammatory markers, and changes in the lung microbiota.

Sa1681 Inhibition of the Prostaglandin EP1 Receptor Suppresses Colon Cancer Cell Invasion and Metastasis.

Background: Kruppel-like factor 5 (KLF5) is a zinc-finger transcription factor that is highly expressed in the crypt cells of the intestinal epithelia and plays a critical role in regulating proliferation of both normal intestinal epithelial cells and colorectal cancer cells. We previously showed that KLF5 is a critical mediator for intestinal tumorigenesis resulted from either ApcMin mutation or oncogenic KRAS activation (McConnell et al. [2009] Cancer Res. 69:4125, Nandan et al. [2010] Molecular Cancer 9:63, respectively). KLF5 is regulated at the transcriptional, translational, and post-translational level. Previous studies indicate that the protein kinase GSK3β and E3 ubiquitin ligase FBW7 facilitate proteosomal degradation of KLF5 protein via phosphorylation (GSK3β) and recognition (FBW7) of a phosphodegron sequence surrounding Ser303 in human KLF5. A recent genomic analysis of colorectal cancer tissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence (Sjoblom et al. [2006] Science 314:268). Aim: To investigate the impact of P301S mutation on KLF5 protein stability. Methods: Plasmids containing human wildtype (WT) and mutant (S303A, P301S, P301A, and P301E) KLF5 under the control of the CMV promoter were transiently transfected into HEK293 cells. The effect of FBW7 on the stability of KLF5 produced from the various constructs was investigated by cycloheximide chase and Western blot analysis. Transcriptional activity of various KLF5 constructs was examined by luciferase reporter assay with Cyclin D1 and CDC2 reporter plasmids. Results: All four KLF5 mutants examined are more resistant to FBW7-mediated proteosomal degradation as compared to WT KLF5. Proteasome inhibition using MG132 resulted in the accumulation of WT KLF5 but had little impact on P301S KLF5 protein levels. Co-immunoprecipitation experiments show that, in contrast to WT KLF5, P301S and S303A KLF5 do not interact with FBW7. Concomitantly, the P301S KLF5 mutant displayed reduced levels of phosphorylation at Ser303 in comparison with WT KLF5. Furthermore, luciferase assays using Cyclin D1 and CDC2 promoter constructs demonstrate significantly elevated transcriptional activity of P301S KLF5 as compared to WT KLF5. Conclusion: Our data indicate that residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence. Furthermore, we show that the colon cancer-derived P301S mutation in KLF5 inhibits FBW7 recognition of the phosphodegron, yielding a degradation-resistant KLF5 protein, elevated protein levels, and enhanced KLF5 transcriptional activity. These results suggest that the P301S mutation in KLF5 is potentially oncogenic in colon cancer.