Prostaglandin E2 in the Tumor Microenvironment, a Convoluted Affair Mediated by EP Receptors 2 and 4.

The involvement of the prostaglandin E2 (PGE2) system in cancer progression has long been recognized. PGE2 functions as an autocrine and paracrine signaling molecule with pleiotropic effects in the human body. High levels of intratumoral PGE2 and overexpression of the key metabolic enzymes of PGE2 have been observed and suggested to contribute to tumor progression. This has been claimed for different types of solid tumors, including, but not limited to, lung, breast, and colon cancer. PGE2 has direct effects on tumor cells and angiogenesis that are known to promote tumor development. However, one of the main mechanisms behind PGE2 driving cancerogenesis is currently thought to be anchored in suppressed antitumor immunity, thus providing possible therapeutic targets to be used in cancer immunotherapies. EP2 and EP4, two receptors for PGE2, are emerging as being the most relevant for this purpose. This review aims to summarize the known roles of PGE2 in the immune system and its functions within the tumor microenvironment. SIGNIFICANCE STATEMENT: Prostaglandin E2 (PGE2) has long been known to be a signaling molecule in cancer. Its presence in tumors has been repeatedly associated with disease progression. Elucidation of its effects on immunological components of the tumor microenvironment has highlighted the potential of PGE2 receptor antagonists in cancer treatment, particularly in combination with immune checkpoint inhibitor therapeutics. Adjuvant treatment could increase the response rates and the efficacy of immune-based therapies.

Cellular senescence and metabolic reprogramming model based on bulk/single-cell RNA sequencing reveals PTGER4 as a therapeutic target for ccRCC.

Clear cell renal cell carcinoma (ccRCC) is the prevailing histological subtype of renal cell carcinoma and has unique metabolic reprogramming during its occurrence and development. Cell senescence is one of the newly identified tumor characteristics. However, there is a dearth of methodical and all-encompassing investigations regarding the correlation between the broad-ranging alterations in metabolic processes associated with aging and ccRCC. We utilized a range of analytical methodologies, such as protein‒protein interaction network analysis and least absolute shrinkage and selection operator (LASSO) regression analysis, to form and validate a risk score model known as the senescence-metabolism-related risk model (SeMRM). Our study demonstrated that SeMRM could more precisely predict the OS of ccRCC patients than the clinical prognostic markers in use. By utilizing two distinct datasets of ccRCC, ICGC-KIRC (the International Cancer Genome Consortium) and GSE29609, as well as a single-cell dataset (GSE156632) and real patient clinical information, and further confirmed the relationship between the senescence-metabolism-related risk score (SeMRS) and ccRCC patient progression. It is worth noting that patients who were classified into different subgroups based on the SeMRS exhibited notable variations in metabolic activity, immune microenvironment, immune cell type transformation, mutant landscape, and drug responsiveness. We also demonstrated that PTGER4, a key gene in SeMRM, regulated ccRCC cell proliferation, lipid levels and the cell cycle in vivo and in vitro. Together, the utilization of SeMRM has the potential to function as a dependable clinical characteristic to increase the accuracy of prognostic assessment for patients diagnosed with ccRCC, thereby facilitating the selection of suitable treatment strategies.

PGE2 limits effector expansion of tumour-infiltrating stem-like CD8+ T cells.

Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1-4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5-9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2-EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.

PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Targeted Gene Deletion or Antagonism of the Prostaglandin E2 EP3 Receptor Protects Against Cardiac Injury Postmyocardial Infarction.

BACKGROUND: Prostaglandin E2 acts through 4 G-protein-coupled receptors (EP1-EP4). We previously reported that activation of the EP3 receptor reduces cardiac contractility, and its expression increases after a myocardial infarction (MI), mediating the reduction in cardiac function. In contrast, cardiac overexpression of the EP4 receptor in MI substantially improves cardiac function. Moreover, we recently reported that mice overexpressing EP3 have heart failure under basal conditions and worsened cardiac function after MI. Thus, the deleterious effects of the prostaglandin E2 EP receptors in the heart are mediated via its EP3 receptor. We, therefore, hypothesized that cardiomyocyte-specific knockout (CM-EP3 KO) or antagonism of the EP3 receptor protects the heart after MI. METHODS: To test our hypothesis, we made the novel CM-EP3 KO mouse and subjected CM-EP3 KO or controls to sham or MI surgery for 2 weeks. In separate experiments, C57BL/6 mice were subjected to 2 weeks of MI and treated with either the EP3 antagonist L798 106 or vehicle starting 3 days post-MI. RESULTS: CM-EP3 KO significantly prevented a decline in cardiac function after MI compared with WT animals and prevented an increase in hypertrophy and fibrosis. Excitingly, mice treated with L798 106 3 days after MI had significantly better cardiac function compared with vehicle-treated mice. CONCLUSIONS: Altogether, these data suggest that EP3 may play a direct role in regulating cardiac function, and pharmaceutical targeting of the EP3 receptor may be a therapeutic option in the treatment of heart failure.

EP2 and EP4 blockade prevents tumor-induced suppressive features in human monocytic myeloid-derived suppressor cells.

Tumors educate their environment to prime the occurrence of suppressive cell subsets, which enable tumor evasion and favors tumor progression. Among these, there are the myeloid-derived suppressor cells (MDSCs), their presence being associated with the poor clinical outcome of cancer patients. Tumor-derived prostaglandin E2 (PGE2) is known to mediate MDSC differentiation and the acquisition of pro-tumor features. In myeloid cells, PGE2 signaling is mediated via E-prostanoid receptor type 2 (EP2) and EP4. Although the suppressive role of PGE2 is well established in MDSCs, the role of EP2/4 on human MDSCs or whether EP2/4 modulation can prevent MDSCs suppressive features upon exposure to tumor-derived PGE2 is poorly defined. In this study, using an in vitro model of human monocytic-MDSCs (M-MDSCs) we demonstrate that EP2 and EP4 signaling contribute to the induction of a pro-tumor phenotype and function on M-MDSCs. PGE2 signaling via EP2 and EP4 boosted M-MDSC ability to suppress T and NK cell responses. Combined EP2/4 blockade on M-MDSCs during PGE2 exposure prevented the occurrence of these suppressive features. Additionally, EP2/4 blockade attenuated the suppressive phenotype of M-MDSCs in a 3D coculture with colorectal cancer patient-derived organoids. Together, these results identify the role of tumor-derived PGE2 signaling via EP2 and EP4 in this human M-MDSC model, supporting the therapeutic value of targeting PGE2-EP2/4 axis in M-MDSCs to alleviate immunosuppression and facilitate the development of anti-tumor immunity.

Prostaglandin E2 accumulation is closely associated with S. aureus-infected bovine endometritis.

S. aureus isolated from bacterial bovine endometritis is common in epidemiological reports, but is often ignored as a subclinical pathogenic microorganism. In a previous study, we showed that live S. aureus (LSA) and heat killed S. aureus (HK-SA) induce different inflammatory responses in bovine endometrial tissue, and possibly being associated with the accumulation of prostaglandin E2 (PGE2). Thus, in this study, we varied PGE2 concentrations using inhibitors or agonists in HK-SA-treated bovine endometrial tissues. The results demonstrated that PGE2 has a positive relationship with IL-6, TNF-α, and damage-associated molecular patterns (DAMPs; e.g., HMGB-1 and HABP-1) expression and tissues damage, and is regulated by the EP4-p38 MAPK pathway. We concluded that lipoproteins of S. aureus are associated with PGE2 generation. To further explore the relationship between LSA and PGE2 accumulation, we used the S. aureus strain SA113 lipoprotein knockout (SA113Δlpl) to infect bovine endometrial epithelial cells (BECs). LSA decreased PGE2, cAMP, EP4, IL-6, IL-8, cAMP secretion, and the MAPK and PKA signaling pathways when infected with SA113Δlpl, as compared with SA113-infected groups. Moreover, the adhesion and invasion of BECs were similarly downregulated when lipoproteins in S. aureus were knocked out. The results of this study show that PGE2 is involved in both HK-SA- and LSA-induced inflammatory responses in the bovine endometrium. We suggest that S. aureus infection is associated with bovine endometritis, and although HK-SA and LSA induce different inflammatory responses, the strategy of decreasing PGE2 accumulation is helpful in reducing the inflammation stage caused by S. aureus.

PGE2-EP4 signaling steers cDC2 maturation toward the induction of suppressive T-cell responses.

Dendritic cells (DCs) shape adaptive immunity in response to environmental cues such as cytokines or lipid mediators, including prostaglandin E2 (PGE2). In cancer, tumors are known to establish an enriched PGE2 microenvironment. Tumor-derived PGE2 primes regulatory features across immune cells, including DCs, facilitating tumor progression. PGE2 shapes DC function by providing signaling via its two so-called E-prostanoid receptors (EPs) EP2 and EP4. Although studies with monocyte-derived DCs have shown the importance of PGE2 signaling, the role of PGE2-EP2/EP4 on conventional DCs type 2 (cDC2s), is still poorly defined. In this study, we investigated the function of EP2 and EP4 using specific EP antagonists on human cDC2s. Our results show that EP2 and EP4 exhibit different functions in cDC2s, with EP4 modulating the upregulation of activation markers (CD80, CD86, CD83, MHC class II) and the production of IL-10 and IL-23. Furthermore, PGE2-EP4 boosts CCR type 7-based migration as well as a higher T-cell expansion capacity, characterized by the enrichment of suppressive rather than pro-inflammatory T-cell populations. Our findings are relevant to further understanding the role of EP receptors in cDC2s, underscoring the benefit of targeting the PGE2-EP2/4 axis for therapeutic purposes in diseases such as cancer.

Design of Dual EP2/EP4 Antagonists through Scaffold Merging of Selective Inhibitors.

Prostaglandin E2 (PGE2) plays a key role in various stages of cancer. PGE2 signals through the EP2 and the EP4 receptors, promoting tumorigenesis, metastasis, and/or immune suppression. Dual inhibition of both the EP2 and the EP4 receptors has the potential to counteract the effect of PGE2 and to result in antitumor efficacy. We herein disclose for the first time the structure of dual EP2/EP4 antagonists. By merging the scaffolds of EP2 selective and EP4 selective inhibitors, we generated a new chemical series of compounds blocking both receptors with comparable potency. In vitro and in vivo profiling suggests that the newly identified compounds are promising lead structures for further development into dual EP2/EP4 antagonists for use in cancer therapy.

Association of PTGER4 and PRKAA1 genetic polymorphisms with gastric cancer.

BACKGROUND: Gastric cancer (GC) is one of the most common malignancies, affected by several genetic loci in the clinical phenotype. This study aimed to determine the association between PTGER4 and PRKAA1 gene polymorphisms and the risk of GC. METHODS: A total of 509 GC patients and 507 age and sex-matched healthy controls were recruited to explore the association between PTGER4 and PRKAA1 genetic polymorphisms and GC susceptibility. Logistic regression analysis was used to study the correlation between these SNPs and GC, with odd ratio (OR) and 95% confidence interval (CI) as indicators. Multifactor dimensionality reduction was utilized to analyze the genetic relationships among SNPs. was conducted to predict gene expression, the impact of SNPs on gene expression, and the signaling pathways involved in PTGER4 and PRKAA1. RESULTS: Overall, rs10036575 in PTGER4 (OR = 0.82, p = 0.029), rs10074991 (OR = 0.82, p = 0.024) and rs13361707 (OR = 0.82, p = 0.030) in PRKAA1 were associated with susceptibility to GC. Stratification analysis revealed that the effects of these SNPs in PTGER4 and PRKAA1 on GC susceptibility were dependent on smoking and were associated with a reduced risk of adenocarcinoma (p < 0.05). Bioinformatics analysis showed an association between SNPs and corresponding gene expression (p < 0.05), and PRKAA1 may affect GC by mediating RhoA. CONCLUSION: This study suggests that PTGER4 and PRKAA1 SNPs might affect the susceptibility of GC, providing a new biological perspective for GC risk assessment, pathogenesis exploration, and personalized treatment.

Dual Blockade of EP2 and EP4 Signaling is Required for Optimal Immune Activation and Antitumor Activity Against Prostaglandin-Expressing Tumors.

While the role of prostaglandin E2 (PGE2) in promoting malignant progression is well established, how to optimally block the activity of PGE2 signaling remains to be demonstrated. Clinical trials with prostaglandin pathway targeted agents have shown activity but without sufficient significance or dose-limiting toxicities that have prevented approval. PGE2 signals through four receptors (EP1-4) to modulate tumor progression. EP2 and EP4 signaling exacerbates tumor pathology and is immunosuppressive through potentiating cAMP production. EP1 and EP3 signaling has the opposite effect through increasing IP3 and decreasing cAMP. Using available small-molecule antagonists of single EP receptors, the cyclooxygenase-2 (COX-2) inhibitor celecoxib, or a novel dual EP2/EP4 antagonist generated in this investigation, we tested which approach to block PGE2 signaling optimally restored immunologic activity in mouse and human immune cells and antitumor activity in syngeneic, spontaneous, and xenograft tumor models. We found that dual antagonism of EP2 and EP4 together significantly enhanced the activation of PGE2-suppressed mouse and human monocytes and CD8+ T cells in vitro as compared with single EP antagonists. CD8+ T-cell activation was dampened by single EP1 and EP3 antagonists. Dual EP2/EP4 PGE2 receptor antagonists increased tumor microenvironment lymphocyte infiltration and significantly reduced disease burden in multiple tumor models, including in the adenomatous polyposis coli (APC)min+/- spontaneous colorectal tumor model, compared with celecoxib. These results support a hypothesis that redundancy of EP2 and EP4 receptor signaling necessitates a therapeutic strategy of dual blockade of EP2 and EP4. Here we describe TPST-1495, a first-in-class orally available small-molecule dual EP2/EP4 antagonist. Significance: Prostaglandin (PGE2) drives tumor progression but the pathway has not been effectively drugged. We demonstrate significantly enhanced immunologic potency and antitumor activity through blockade of EP2 and EP4 PGE2 receptor signaling together with a single molecule.

Prorelaxant E-type Prostanoid Receptors Functionally Partition to Different Procontractile Receptors in Airway Smooth Muscle.

Prostaglandin E2 imparts diverse physiological effects on multiple airway cells through its actions on four distinct E-type prostanoid (EP) receptor subtypes (EP1-EP4). Gs-coupled EP2 and EP4 receptors are expressed on airway smooth muscle (ASM), yet their capacity to regulate the ASM contractile state remains subject to debate. We used EP2 and EP4 subtype-specific agonists (ONO-259 and ONO-329, respectively) in cell- and tissue-based models of human ASM contraction-magnetic twisting cytometry (MTC), and precision-cut lung slices (PCLSs), respectively-to study the EP2 and EP4 regulation of ASM contraction and signaling under conditions of histamine or methacholine (MCh) stimulation. ONO-329 was superior (<0.05) to ONO-259 in relaxing MCh-contracted PCLSs (log half maximal effective concentration [logEC50]: 4.9 × 10-7 vs. 2.2 × 10-6; maximal bronchodilation ± SE, 35 ± 2% vs. 15 ± 2%). However, ONO-259 and ONO-329 were similarly efficacious in relaxing histamine-contracted PCLSs. Similar differential effects were observed in MTC studies. Signaling analyses revealed only modest differences in ONO-329- and ONO-259-induced phosphorylation of the protein kinase A substrates VASP and HSP20, with concomitant stimulation with MCh or histamine. Conversely, ONO-259 failed to inhibit MCh-induced phosphorylation of the regulatory myosin light chain (pMLC20) and the F-actin/G-actin ratio (F/G-actin ratio) while effectively inhibiting their induction by histamine. ONO-329 was effective in reversing induced pMLC20 and the F/G-actin ratio with both MCh and histamine. Thus, the contractile-agonist-dependent differential effects are not explained by changes in the global levels of phosphorylated protein kinase A substrates but are reflected in the regulation of pMLC20 (cross-bridge cycling) and F/G-actin ratio (actin cytoskeleton integrity, force transmission), implicating a role for compartmentalized signaling involving muscarinic, histamine, and EP receptor subtypes.

PGE2-EP2/EP4 signaling elicits mesoCAR T cell immunosuppression in pancreatic cancer.

Introduction: For many years, surgery, adjuvant and combination chemotherapy have been the cornerstone of pancreatic cancer treatment. Although these approaches have improved patient survival, relapse remains a common occurrence, necessitating the exploration of novel therapeutic strategies. CAR T cell therapies are now showing tremendous success in hematological cancers. However, the clinical efficacy of CAR T cells in solid tumors remained low, notably due to presence of an immunosuppressive tumor microenvironment (TME). Prostaglandin E2, a bioactive lipid metabolite found within the TME, plays a significant role in promoting cancer progression by increasing tumor proliferation, improving angiogenesis, and impairing immune cell's function. Despite the well-established impact of PGE2 signaling on cancer, its specific effects on CAR T cell therapy remain under investigation. Methods: To address this gap in knowledge the role of PGE2-related genes in cancer tissue and T cells of pancreatic cancer patients were evaluated in-silico. Through our in vitro study, we manufactured fully human functional mesoCAR T cells specific for pancreatic cancer and investigated the influence of PGE2-EP2/EP4 signaling on proliferation, cytotoxicity, and cytokine production of mesoCAR T cells against pancreatic cancer cells. Results: In-silico investigations uncovered a significant negative correlation between PGE2 expression and gene signature of memory T cells. Furthermore, in vitro experiments demonstrated that the activation of PGE2 signaling through EP2 and EP4 receptors suppressed the proliferation and major antitumor functions of mesoCAR T cells. Interestingly, the dual blockade of EP2 and EP4 receptors effectively reversed PGE2-mediated suppression of mesoCAR T cells, while individual receptor antagonists failed to mitigate the PGE2-induced suppression. Discussion: In summary, our findings suggest that mitigating PGE2-EP2/EP4 signaling may be a viable strategy for enhancing CAR T cell activity within the challenging TME, thereby improving the efficacy of CAR T cell therapy in clinical settings.

Complementary roles of EP2 and EP4 receptors in malignant glioma.

BACKGROUND AND PURPOSE: Glioblastoma (GBM) is the most aggressive brain tumour in the central nervous system, but the current treatment is very limited and unsatisfactory. PGE2 -initiated cAMP signalling via EP2 and EP4 receptors is involved in the tumourigenesis of multiple cancer types. However, whether or how EP2 and EP4 receptors contribute to GBM growth largely remains elusive. EXPERIMENTAL APPROACH: We performed comprehensive data analysis of gene expression in human GBM samples and determined their expression correlations through multiple bioinformatics approaches. A time-resolved fluorescence energy transfer (TR-FRET) assay was utilized to characterize PGE2 -mediated cAMP signalling via EP2 and EP4 receptors in human glioblastoma cells. Using recently reported potent and selective small-molecule antagonists, we determined the effects of inhibition of EP2 and EP4 receptors on GBM growth in subcutaneous and intracranial tumour models. KEY RESULTS: The expression of both EP2 and EP4 receptors was upregulated and highly correlated with a variety of tumour-promoting cytokines, chemokines, and growth factors in human gliomas. Further, they were heterogeneously expressed in human GBM cells, where they compensated for each other to mediate PGE2 -initiated cAMP signalling and to promote colony formation, cell invasion and migration. Inhibition of EP2 and EP4 receptors revealed that these receptors might mediate GBM growth, angiogenesis, and immune evasion in a compensatory manner. CONCLUSION AND IMPLICATIONS: The compensatory roles of EP2 and EP4 receptors in GBM development and growth suggest that concurrently targeting these two PGE2 receptors might represent a more effective strategy than inhibiting either alone for GBM treatment.

Discovery of 2H-Indazole-3-carboxamide Derivatives as Novel Potent Prostanoid EP4 Receptor Antagonists for Colorectal Cancer Immunotherapy.

Nowadays, small-molecule drugs have become an indispensable part of tumor immunotherapy. Accumulating evidence has indicated that specifically blocking PGE2/EP4 signaling to induce robust antitumor immune response represents an attractive immunotherapy strategy. Herein, a 2H-indazole-3-carboxamide containing compound 1 was identified as a EP4 antagonist hit by screening our in-house small-molecule library. Systematic structure-activity relationship exploration leads to the discovery of compound 14, which displayed single-nanomolar EP4 antagonistic activity in a panel of cell functional assays, high subtype selectivity, and favorable drug-like profiles. Moreover, compound 14 profoundly inhibited the up-regulation of multiple immunosuppression-related genes in macrophages. Oral administration of compound 14, either as monotherapy or in combination with an anti-PD-1 antibody, significantly impaired tumor growth via enhancing cytotoxic CD8+ T cell-mediated antitumor immunity in a syngeneic colon cancer model. Thus, these results demonstrate the potential of compound 14 as a candidate for developing novel EP4 antagonists for tumor immunotherapy.

Prostaglandin E2-Transporting Pathway and Its Roles via EP2/EP4 in Cultured Human Dental Pulp.

INTRODUCTION: Prostaglandin E2 (PGE2) exerts biological actions through its transport pathway involving intracellular synthesis, extracellular transport, and receptor binding. This study aimed to determine the localization of the components of the PGE2-transporting pathway in human dental pulp and explore the relevance of PGE2 receptors (EP2/EP4) to angiogenesis and dentinogenesis. METHODS: Protein localization of microsomal PGE2 (mPGES)synthase, PGE2 transporters (multidrug resistance-associated protein-4 [MRP4] and prostaglandin transporter [PGT]), and EP2/EP4 was analyzed using double immunofluorescence staining. Tooth slices from human third molars were cultured with or without butaprost (EP2 agonist) or rivenprost (EP4 agonist) for 1 week. Morphometric analysis of endothelial cell filopodia was performed to evaluate angiogenesis, and real-time polymerase chain reaction was performed to evaluate angiogenesis and odontoblast differentiation markers. RESULTS: MRP4 and PGT were colocalized with mPGES and EP2/EP4 in odontoblasts and endothelial cells. Furthermore, MRP4 was colocalized with mPGES and EP4 in human leukocyte antigen-DR-expressing dendritic cells. In the tooth slice culture, EP2/EP4 agonists induced significant increases in the number and length of filopodia and mRNA expression of angiogenesis markers (vascular endothelial growth factor and fibroblast growth factor-2) and odontoblast differentiation markers (dentin sialophosphoprotein and collagen type 1). CONCLUSIONS: PGE2-producing enzyme (mPGES), transporters (MRP4 and PGT), and PGE2-specific receptors (EP2/EP4) were immunolocalized in various cellular components of the human dental pulp. EP2/EP4 agonists promoted endothelial cell filopodia generation and upregulated angiogenesis- and odontoblast differentiation-related genes, suggesting that PGE2 binding to EP2/EP4 is associated with angiogenic and dentinogenic responses.

Tailored PGE2 Immunomodulation of moDCs by Nano-Encapsulated EP2/EP4 Antagonists.

Prostaglandin E2 (PGE2) is an important maturation mediator for dendritic cells (DCs). However, increased PGE2 levels in the tumor exert immunosuppressive effects on DCs by signaling through two E-Prostanoid (EP) receptors: EP2 and EP4. Blocking EP-receptor signaling of PGE2 with antagonists is currently being investigated for clinical applications to enhance anti-tumor immunity. In this study, we investigated a new delivery approach by encapsulating EP2/EP4 antagonists in polymeric nanoparticles. The nanoparticles were characterized for size, antagonist loading, and release. The efficacy of the encapsulated antagonists to block PGE2 signaling was analyzed using monocyte-derived DCs (moDCs). The obtained nanoparticles were sized between 210 and 260 nm. The encapsulation efficacy of the EP2/EP4 antagonists was 20% and 17%, respectively, and was further increased with the co-encapsulation of both antagonists. The treatment of moDCs with co-encapsulation EP2/EP4 antagonists prevented PGE2-induced co-stimulatory marker expression. Even though both antagonists showed a burst release within 15 min at 37 °C, the nanoparticles executed the immunomodulatory effects on moDCs. In summary, we demonstrate the functionality of EP2/EP4 antagonist-loaded nanoparticles to overcome PGE2 modulation of moDCs.

Prostaglandin EP3 receptor activation is antinociceptive in sensory neurons via PI3Kγ, AMPK and GRK2.

BACKGROUND AND PURPOSE: Prostaglandin E2 is considered a major mediator of inflammatory pain, by acting on neuronal Gs protein-coupled EP2 and EP4 receptors. However, the neuronal EP3 receptor, colocalized with EP2 and EP4 receptor, is Gi protein-coupled and antagonizes the pronociceptive prostaglandin E2 effect. Here, we investigated the cellular signalling mechanisms by which the EP3 receptor reduces EP2 and EP4 receptor-evoked pronociceptive effects in sensory neurons. EXPERIMENTAL APPROACH: Experiments were performed on isolated and cultured dorsal root ganglion (DRG) neurons from wild type, phosphoinositide 3-kinase γ (PI3Kγ)-/- , and PI3Kγkinase dead (KD)/KD mice. For subtype-specific stimulations, we used specific EP2, EP3, and EP4 receptor agonists from ONO Pharmaceuticals. As a functional readout, we recorded TTX-resistant sodium currents in patch-clamp experiments. Western blots were used to investigate the activation of intracellular signalling pathways. EP4 receptor internalization was measured using immunocytochemistry. KEY RESULTS: Different pathways mediate the inhibition of EP2 and EP4 receptor-dependent pronociceptive effects by EP3 receptor stimulation. Inhibition of EP2 receptor-evoked pronociceptive effect critically depends on the kinase-independent function of the signalling protein PI3Kγ, and adenosine monophosphate activated protein kinase (AMPK) is involved. By contrast, inhibition of EP4 receptor-evoked pronociceptive effect is independent on PI3Kγ and mediated through activation of G protein-coupled receptor kinase 2 (GRK2), which enhances the internalization of the EP4 receptor after ligand binding. CONCLUSION AND IMPLICATIONS: Activation of neuronal PI3Kγ, AMPK, and GRK2 by EP3 receptor activation limits cAMP-dependent pain generation by prostaglandin E2 . These new insights hold the potential for a novel approach in pain therapy.

Epithelial presenilin-1 drives colorectal tumour growth by controlling EGFR-COX2 signalling.

OBJECTIVE: Psen1 was previously characterised as a crucial factor in the pathogenesis of neurodegeneration in patients with Alzheimer's disease. Little, if any, is known about its function in the gut. Here, we uncovered an unexpected functional role of Psen1 in gut epithelial cells during intestinal tumourigenesis. DESIGN: Human colorectal cancer (CRC) and control samples were investigated for PSEN1 and proteins of theγ-secretase complex. Tumour formation was analysed in the AOM-DSS and Apc min/+ mouse models using newly generated epithelial-specific Psen1 deficient mice. Psen1 deficient human CRC cells were studied in a xenograft tumour model. Tumour-derived organoids were analysed for growth and RNA-Seq was performed to identify Psen1-regulated pathways. Tumouroids were generated to study EGFR activation and evaluation of the influence of prostanoids. RESULTS: PSEN1 is expressed in the intestinal epithelium and its level is increased in human CRC. Psen1-deficient mice developed only small tumours and human cancer cell lines deficient in Psen1 had a reduced tumourigenicity. Tumouroids derived from Psen1-deficient Apc min/+ mice exhibited stunted growth and reduced cell proliferation. On a molecular level, PSEN1 potentiated tumour cell proliferation via enhanced EGFR signalling and COX-2 production. Exogenous administration of PGE2 reversed the slow growth of PSEN1 deficient tumour cells via PGE2 receptor 4 (EP4) receptor signalling. CONCLUSIONS: Psen1 drives tumour development by increasing EGFR signalling via NOTCH1 processing, and by activating the COX-2-PGE2 pathway. PSEN1 inhibition could be a useful strategy in treatment of CRC.

First-in-human study of ONO-4578, an antagonist of prostaglandin E2 receptor 4, alone and with nivolumab in solid tumors.

EP4, a prostaglandin E2 receptor, has shown an immunosuppressive activity on cancer cells. This first-in-human study evaluated ONO-4578, a highly selective EP4 antagonist, as monotherapy and in combination with nivolumab in patients with advanced or metastatic solid tumors. A daily dose ranging from 30 mg to 100 mg of ONO-4578 monotherapy and that ranging from 2 mg to 60 mg of ONO-4578 with biweekly nivolumab 240 mg were administered. A total of 31 patients were enrolled, 10 receiving monotherapy and 21 receiving combination therapy. Overall, 26 patients experienced treatment-related adverse events. Dose-limiting toxicities were observed in three patients; one of six patients receiving 100 mg monotherapy developed grade 3 duodenal ulcer and two of six patients receiving 60 mg combination therapy developed either grade 3 erythema multiforme or grade 3 increased amylase and grade 4 increased lipase. One patient with small-cell lung cancer who received 40 mg combination therapy had a partial response, and three patients with monotherapy and six patients with combination therapy had stable disease. Pharmacodynamics analyses showed that ONO-4578 had EP4 antagonistic activity at doses as low as 2 mg. In conclusion, the maximum tolerated dose of ONO-4578 alone or in combination with nivolumab was not reached. ONO-4578 was well tolerated at the tested doses and showed signs of antitumor activity. Considering safety, efficacy, and pharmacokinetics/pharmacodynamics results, ONO-4578 40 mg daily with nivolumab 240 mg biweekly was selected as the recommended dose for future clinical trials. (Registration: JapicCTI-173,496 and NCT03155061).