EP4 Receptor Research Articles

Effects of NP-1815-PX, a P2X4 Receptor Antagonist, on Contractions in Guinea Pig Tracheal and Bronchial Smooth Muscles.

Administration of a P2X4 receptor antagonist to asthma model mice improved asthma symptoms, suggesting that P2X4 receptor antagonists may be new therapeutics for asthma. However, the effects of these antagonists on tracheal/bronchial smooth muscle (TSM and BSM) have not been investigated. This study examined the effects of NP-1815-PX, a selective P2X4 receptor antagonist, on guinea pig TSM and BSM contractions. In epithelium-intact TSM, NP-1815-PX (10-5 M) strongly suppressed ATP-induced contractions. ATP-induced contractions were strongly suppressed by indomethacin (3 × 10-6 M) and ONO-8130 (a prostanoid EP1 receptor antagonist, 10-7 M). ATP-induced contractions were partially suppressed by SQ 29,548 (a prostanoid TP receptor antagonist, 3 × 10-7 M), although the difference was not significant. In contrast, ATP-induced contractions were not affected by AL 8810 (a prostanoid FP receptor antagonist, 10-5 M) or L-798,106 (a prostanoid EP3 receptor antagonist, 10-8 M). NP-1815-PX (10-5-10-4 M) strongly suppressed U46619 (a TP receptor agonist)- and prostaglandin F2α (PGF2α)-induced epithelium-denuded TSM and BSM contractions, which were largely inhibited by SQ 29,548. Additionally, NP-1815-PX (10-5-10-4 M) strongly suppressed the U46619-induced increase in intracellular Ca2+ concentrations in human TP receptor-expressing cells. However, NP-1815-PX (10-4 M) did not substantially inhibit the TSM/BSM contractions induced by carbachol, histamine, neurokinin A, or 50 mM KCl. These findings indicate that NP-1815-PX inhibits guinea pig TSM and BSM contractions mediated through the TP receptor, in addition to the P2X4 receptor, whose stimulation mainly induces EP1 receptor-related mechanisms. Thus, these findings support the usefulness of NP-1815-PX as a therapeutic drug for asthma.

Lung fibroblasts facilitate pre-metastatic niche formation by remodeling the local immune microenvironment

Primary tumors are drivers of pre-metastatic niche formation, but the coordination by the secondary organ toward metastatic dissemination is underappreciated. Here, by single-cell RNA sequencing and immunofluorescence, we identified a population of cyclooxygenase 2 (COX-2)-expressing adventitial fibroblasts that remodeled the lung immune microenvironment. At steady state, fibroblasts in the lungs produced prostaglandin E2 (PGE2), which drove dysfunctional dendritic cells (DCs) and suppressive monocytes. This lung-intrinsic stromal program was propagated by tumor-associated inflammation, particularly the pro-inflammatory cytokine interleukin-1β, supporting a pre-metastatic niche. Genetic ablation of Ptgs2 (encoding COX-2) in fibroblasts was sufficient to reverse the immune-suppressive phenotypes of lung-resident myeloid cells, resulting in heightened immune activation and diminished lung metastasis in multiple breast cancer models. Moreover, the anti-metastatic activity of DC-based therapy and PD-1 blockade was improved by fibroblast-specific Ptgs2 deletion or dual inhibition of PGE2 receptors EP2 and EP4. Collectively, lung-resident fibroblasts reshape the local immune landscape to facilitate breast cancer metastasis.

Neurokinin 1 and 2 receptors are involved in PGE2- and citric acid-induced cough and ventilatory responses

Exposure to aerosolized citric acid (CA, 150 mM) and prostaglandin E2 (PGE2, 0.43 mM) for 10 min in guinea pigs reportedly produces the distinct cough patterns (Type I vs. II) and ventilatory responses (long-lasting hyperventilation vs. brief tachypnea) even though triggering the same cough numbers. Type I and II coughs are primarily mediated by activation of TRPV1 and EP3 receptors (a PGE2 receptor) of vagal C-fibers respectively. Substance P (SP) and neurokinin A (NKA) released by vagal pulmonary sensory fibers peripherally are capable of affecting CA-induced cough and ventilation via preferentially activating neurokinin 1 and 2 receptors (NK1R and NK2R) respectively. This study aimed to define the impacts of CA- and PGE2-exposure on pulmonary SP and NKA levels and the roles of NK1R and NK2R in modulating CA- and PGE2-evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents induced by the CA- or PGE2-exposure; (2) effects of CP-99994 and SR-48968 (a NK1R and a NK2R antagonist respectively) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on the CA- and PGE2-evoked cough and ventilatory responses; and (3) immunocytochemical expressions of NK1R/NK2R in vagal C-neurons labeled by TRPV1 or EP3 receptors. We found that CA- and PGE2-exposure evoked Type I and II cough respectively associated with different degrees of increases in pulmonary SP and NKA. Applications of CP-99994 and SR-48968 via IP and IH efficiently suppressed the cough responses to CA with less impact on the cough response to PGE2. These antagonists inhibited or blocked the ventilatory response to CA and caused hypoventilation in response to PGE2. Moreover, NK1R and NK2R were always co-expressed in vagal C-neurons labeled by TRPV1 or EP3 receptors. These results suggest that SP and NKA endogenously released by CA- and PGE2-exposure play important roles in generating the cough and ventilatory responses to CA and PGE2, at least in part, via activation of NK1R and NK2R expressed in vagal C-neurons (pulmonary C-neurons).

Platelet-derived growth factor (PDGF)-BB regulates the airway tone via activation of MAP2K, thromboxane, actin polymerisation and Ca2+-sensitisation

BackgroundPDGFR-inhibition by the tyrosine kinase inhibitor (TKI) nintedanib attenuates the progress of idiopathic pulmonary fibrosis (IPF). However, the effects of PDGF-BB on the airway tone are almost unknown. We studied this issue and the mechanisms beyond, using isolated perfused lungs (IPL) of guinea pigs (GPs) and precision-cut lung slices (PCLS) of GPs and humans.MethodsIPL: PDGF-BB was perfused after or without pre-treatment with the TKI imatinib (perfused/nebulised) and its effects on the tidal volume (TV), the dynamic compliance (Cdyn) and the resistance were studied. PCLS (GP): The bronchoconstrictive effects of PDGF-BB and the mechanisms beyond were evaluated. PCLS (human): The bronchoconstrictive effects of PDGF-BB and the bronchorelaxant effects of imatinib were studied. All changes of the airway tone were measured by videomicroscopy and indicated as changes of the initial airway area.ResultsPCLS (GP/human): PDGF-BB lead to a contraction of airways. IPL: PDGF-BB decreased TV and Cdyn, whereas the resistance did not increase significantly. In both models, inhibition of PDGFR-(β) (imatinib/SU6668) prevented the bronchoconstrictive effect of PDGF-BB. The mechanisms beyond PDGF-BB-induced bronchoconstriction include activation of MAP2K and TP-receptors, actin polymerisation and Ca2+-sensitisation, whereas the increase of Ca2+ itself and the activation of EP1–4-receptors were not of relevance. In addition, imatinib relaxed pre-constricted human airways.ConclusionsPDGFR regulates the airway tone. In PCLS from GPs, this regulatory mechanism depends on the β-subunit. Hence, PDGFR-inhibition may not only represent a target to improve chronic airway disease such as IPF, but may also provide acute bronchodilation in asthma. Since asthma therapy uses topical application. This is even more relevant, as nebulisation of imatinib also appears to be effective.

The Succinate Receptor SUCNR1 Resides at the Endoplasmic Reticulum and Relocates to the Plasma Membrane in Hypoxic Conditions.

The GPCR SUCNR1/GPR91 exerts proangiogenesis upon stimulation with the Krebs cycle metabolite succinate. GPCR signaling depends on the surrounding environment and intracellular localization through location bias. Here, we show by microscopy and by cell fractionation that in neurons, SUCNR1 resides at the endoplasmic reticulum (ER), while being fully functional, as shown by calcium release and the induction of the expression of the proangiogenic gene for VEGFA. ER localization was found to depend upon N-glycosylation, particularly at position N8; the nonglycosylated mutant receptor localizes at the plasma membrane shuttled by RAB11. This SUCNR1 glycosylation is physiologically regulated, so that during hypoxic conditions, SUCNR1 is deglycosylated and relocates to the plasma membrane. Downstream signal transduction of SUCNR1 was found to activate the prostaglandin synthesis pathway through direct interaction with COX-2 at the ER; pharmacologic antagonism of the PGE2 EP4 receptor (localized at the nucleus) was found to prevent VEGFA expression. Concordantly, restoring the expression of SUCNR1 in the retina of SUCNR1-null mice renormalized vascularization; this effect is markedly diminished after transfection of the plasma membrane-localized SUCNR1 N8A mutant, emphasizing that ER localization of the succinate receptor is necessary for proper vascularization. These findings uncover an unprecedented physiologic process where GPCR resides at the ER for signaling function.

Losac and Lopap Recombinant Proteins from Lonomia obliqua Bristles Positively Modulate the Myoblast Proliferation Process.

The pursuit of better therapies for disorders creating deficiencies in skeletal muscle regeneration is in progress, and several biotoxins are used in skeletal muscle research. Since recombinant proteins derived from Lonomia obliqua bristles, recombinant Lonomia obliqua Stuart-factor activator (rLosac) and recombinant Lonomia obliqua prothrombin activator protease (rLopap) act as cytoprotective agents and promote cell survival, we hypothesize that both rLosac and rLopap favour the skeletal muscle regeneration process. In the present work, we investigate the ability of these recombinant proteins rLosac and rLopap to modulate the production of key mediators of the myogenic process. The expression of myogenic regulatory factors (MRFs), cell proliferation, the production of prostaglandin E2 (PGE2) and the protein expression of cyclooxygenases COX-1 and COX-2 were evaluated in C2C12 mouse myoblasts pre-treated with rLosac and rLopap. We found an increased proliferation of myoblasts, stimulated by both recombinant proteins. Moreover, these proteins modulated PGE2 release and MRFs activities. We also found an increased expression of the EP4 receptor in the proliferative phase of C2C12 cells, suggesting the involvement of this receptor in the effects of PGE2 in these cells. Moreover, the recombinant proteins inhibited the release of IL-6 and PGE2, which is induced by an inflammatory stimulus by IL-1β. This work reveals rLopap and rLosac as promising proteins to modulate processes involving tissue regeneration as occurs during skeletal muscle injury.

Abstract 1333: Dual blockade of the EP2 and EP4 PGE2 receptors with TPST-1495 is an optimal approach for drugging the prostaglandin pathway

Abstract Prostaglandin E2 (PGE2) is a bioactive lipid produced by tumor cells that drives disease progression through stimulating tumor proliferation, enhancing angiogenesis and suppressing immune function in the TME1, 2. PGE2 is also a mediator of adaptive resistance to immune checkpoint inhibitor therapy via the upregulation of cyclooxygenase-2 (COX-2). While the role of PGE2 signaling in cancer is clear, how best to inhibit PGE2 for cancer treatment remains under investigation. Inhibition of COX-1 and/or COX-2 has shown promising results in observational studies and meta-analyses, but inconsistent results in prospective studies. PGE2 signals through four receptors, EP1-4, that are variably expressed on tumor and immune cells and have distinct biological activities. The EP2 and EP4 receptors signal through cAMP and drive pro-tumor activities, while the EP1 and EP3 receptors signal through calcium flux and IP3 and drive immune activation and inflammation. While COX-2 and single EP inhibitors continue to be developed, the nature of PGE2 signaling supports our rationale to inhibit PGE2 by dual antagonism of the pro-tumor EP2/EP4 receptors, while sparing the pro-immune EP1/EP3 receptors. To our knowledge, TPST-1495 is the first clinical-stage dual inhibitor of both the EP2 and EP4 receptors. In mouse and human whole blood assays, dual blockade of EP2 and EP4 receptors with TPST-1495 reversed PGE2-mediated suppression of LPS-induced TNF-α, while single receptor antagonists were unable to block suppression at higher PGE2 concentrations. Similarly, in murine and human T cells in vitro, TPST-1495 inhibited PGE2-mediated suppression, resulting in a significant increase of IFN-γ production in response to stimulation with cognate peptide antigen. In vivo, TPST-1495 monotherapy significantly reduced tumor outgrowth in CT26 tumor-bearing mice and correlated with increased tumor infiltration by NK cells, CD8+ T cells, AH1-specific CD8+ T cells, and other anti-tumor myeloid and adaptive immune cell populations. The relative contribution of increased immune infiltration may be simultaneously dependent on the immunogenicity of the tumor model and on the direct antitumor effect of TPST-1495, because we also observed significant tumor regression in metastatic burden in the LS174T xenograft model in NSG mice as well as CT26 tumors in RAG2-/- animals, both of which are deficient in immune cell development. To that end, TPST- 1495 monotherapy significantly decreased the tumor burden compared to COX2 inhibition and EP2 or EP4 single antagonism in the Adenomatous Polyposis (APCmin/+) model, a model that is hypo-responsive to PD-1 monotherapy. TPST-1495 is currently being evaluated in an ongoing Phase 1 first-in-human study (NCT04344795) to characterize PK, PD, safety, and to identify a recommended phase 2 dose for expansion cohorts in key indications and biomarker-selected patients. Citation Format: Brian Francica, Justine Lopez, Anja Holtz, Dave Freund, Dingzhi Wang, Amanda Enstrom, Raymond Dubois, Francielle Kipper, Dipak Panigrahy, Chan Whiting, Sam Whiting, Thomas W. Dubensky. Dual blockade of the EP2 and EP4 PGE2 receptors with TPST-1495 is an optimal approach for drugging the prostaglandin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1333.

Abstract 3139: Disrupting prostaglandin E2 signaling to reverse treatment resistance in cancer

Abstract Pancreatic adenocarcinoma (PDA) accounts for 3% of all cancers in the USA and 7% of all cancer deaths partly because of its resistance to immunotherapy. PDA manifests itself as a desmoplastic, myeloid cell-rich tumor with a notable paucity of T cells. Pathway analysis of the T cell low, immunotherapy resistant mouse PDA cell lines has identified the prostaglandin E2 (PGE2) synthetic pathway as one of the most upregulated ones. We have previously shown that the deletion or pharmacological inhibition of COX-2, the rate-limiting PGE2 synthesis enzyme, slows the tumor growth and synergizes with immunotherapy to further suppress PDA progression. The current study investigates how the disruption of PGE2 signaling through its receptors, EP4 and EP2, can affect PDA. We use EP receptor genetic and pharmacological inhibition models - we disrupt EP signaling on tumor cells only or systemically, test the effects in T cell low, intermediate, and high PDA tumors, alone or in combination with chemo and immunotherapy. EP4 receptor blocker combined with the agonistic CD40 and anti-PD-1 antibodies significantly increased tumor-free survival of the T cell intermediate PDA implanted tumor-bearing (TB) mice (57% tumor-free in treated vs 0% in the control group). The same combination therapy tended to increase the survival in autochthonous tumor-bearing KrasG12D and p53R172H transgenic mice (p=0.08). CRISPR knockout (KO) of the EP4 in the T cell low cell line abolished the growth of subcutaneous (SQ) tumors and significantly slowed the progression of orthotopically implanted tumors (p=0.009 EP4 KO vs control). Treatment of the SQ T cell low TB mice with EP2/EP4 blockers in combination with either anti-PD-1 or chemotherapy slowed the tumor growth compared to the controls (p=0.002 and p=0.004, respectively) and increased the survival (p=0.007 and p=0.002, respectively). A week of combination treatment resulted in changes in the myeloid cell compartment of the TME - an increase in the proportion of M1 macrophages and type 1 conventional dendritic cells with no significant changes in the T cell compartment. The study demonstrates an alternative approach to targeting the PGE2 pathway in tumors and aims to develop a multiprong treatment regimen to reverse the therapy resistance in PDA. Normal 0 false false false EN-US X-NONE X-NONE/* Style Definitions */table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;} <![endif]–> Citation Format: Nune Markosyan, Liz Quinones, Charu Aurora, Nikhil Joshi, Ilkyu Kim, Ben Z. Stanger, Robert H. Vonderheide. Disrupting prostaglandin E2 signaling to reverse treatment resistance in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3139.

Mesenchymal Stem Cells Attenuate Acute Lung Injury in Mice Partly by Suppressing Alveolar Macrophage Activation in a PGE2-Dependent Manner.

Mesenchymal stem cells (MSCs) have been demonstrated to attenuate acute lung injury (ALI). We also found that they can suppress the activation of alveolar macrophages (AMs), which can partly account for their therapeutic effects. MSCs do not inherently own immunosuppressive effects, when co-cultured with inflammatory immune cells, MSCs can be activated by inflammatory cytokines and meanwhile exert immunosuppressive effects. In order to further research, RNA sequencing (RNA-seq) of MSCs cultured before and after co-culturing with activated macrophages was performed. The data suggested a total of 5268 differentially expressed genes (DEGs) along the process. We used the data of 2754 upregulated DEGs to develop a signaling network of genes and the transcription factors targeting them in order to predict the altered functions of MSCs after exposure to inflammatory stimuli. This constructed network revealed some critical target genes and potential roles of MSCs under inflammatory conditions. According to the network, Ptgs2 was assumed to be an important gene participating in the immunosuppressive effects of MSCs. We also identified significant increases in the expression of COX2 protein and the secretion of PGE2 from MSCs. The use of the COX2 inhibitor NS-398 restrained the secretion of PGE2 and reversed the suppression of macrophage activation by MSCs in vitro. In addition, a selective antagonist of PGE2 binding receptor (EP4 receptor), GW627368X, also reversed the inhibitory effects of MSCs on AMs and the protective effects in ALI mouse. In summary, the therapeutic effects of MSCs on ALI partly occur through suppressing AM activation via PGE2 binding to EP4 receptor.

Does the Combined Use of Aspirin and Immunotherapy Result in Better Outcomes in Non-Small Cell Lung Cancer Than Immunotherapy Alone?

Introduction: Immunotherapy works by stimulating the immune system against cancer cells. Resistance to immunotherapy represents a significant challenge in the field of medical oncology. The mechanisms by which cancer cells evade immunotherapy are not well understood. Prior research suggested overexpression of prostaglandin E-2 (PGE-2) by cancer cells, which bind to EP-2 and EP-4 receptors on the tumor-specific cytotoxic T-lymphocytes (CTLs) and suppress their anticancer role. This immunosuppressive effect is involved in evading the programmed cell death-1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade of immunotherapy, which fuels cancer cell growth and recurrence. Studies found that combining PGE-2 blockade and a PD-1 signaling inhibitor helped promote the anticancer immunity cells. If confirmed in a clinical setting, the above in vitro findings could be of great clinical significance.Methods: Given that aspirin (ASA) blocks PGE-2 production, this work aimed to evaluate whether ASA use with immunotherapy results in better outcomes than immunotherapy alone. We performed a retrospective chart review of 500 non-small cell lung cancer (NSCLC) patients aged 21 years or older treated with PD-1 and/or PD-L1 directed immunotherapy at St. Luke’s University Health Network between July 2015 and July 2021. Relevant patient, disease, and treatment-related variables were collected, including ASA use (≥ 81 mg daily) and the type of immunotherapy. Bivariate analyses were conducted to determine which variables to include in a multivariable model. The four primary outcomes included survival at 18-months, both after diagnosis and starting immunotherapy, achieving complete remission (CR), and having a progressive disease (PD), as defined by RECIST (Response Evaluation Criteria in Solid Tumors) criteria. Secondary outcomes included therapy-related toxicities and complications in the different treatment groups. Results: After bivariate analysis, no statistical significance was found for a difference in 18-month survival between ASA and non-ASA groups (50.3% vs 49.7%, p-value = 0.79). ASA with PD-L1 inhibitor showed a trend towards a higher likelihood of achieving CR [adjusted odds ratio (AOR) 1.85] with a p-value close to statistical significance (0.06). However, ASA with PD-L1 showed high statistical significance as an independent variable associated with a decreased likelihood of having PD (AOR 0.44, p < 0.001). These findings suggest that NSCLC patients receiving PD-L1 inhibitors could benefit more from daily ASA than patients treated with PD-1 inhibitors. Our study emphasizes using the Eastern Cooperative Oncology Group (ECOG) scoring of the performance status (PS) in NSCLC patients. Poorer PS was associated with lower survival, decreased likelihood of CR, and more PD. Other variables associated with worse outcomes were advanced cancer stage at diagnosis and male gender. Low-PD-L1 expression in NSCLC was associated with an increased likelihood of survival; this could be of clinical significance, especially with previous studies suggesting better outcomes of using ASA in PD-L1 low tumors. Conclusion: These findings suggest that daily ASA use with PD-L1 inhibitors is associated with more favorable outcomes in NSCLC. More studies are needed to investigate further the potential benefits vs. risks of using ASA with different immunotherapies and the other possible variables affecting treatment outcomes.

PGE2 inhibits neutrophil phagocytosis through the EP2R-cAMP-PTEN pathway.

Prostaglandin E2 (PGE2) is a potent lipid mediator of inflammation that modulates immune cell function by binding to unique G protein‐coupled receptors (EP receptors). PGE2 production increases during microbial infection and inflammation. In this study, we assessed the effect of PGE2 on the phagocytosis of bacteria by neutrophils, which are key players during infection and inflammation. We also looked for specific EP receptor signaling pathways that contributed to the neutrophil phagocytic activity. PGE2 (50–1000 ng/ml) inhibited the phagocytosis of Escherichia coli by HL‐60 human neutrophils in a concentration‐dependent manner. Inhibition of neutrophil phagocytosis by PGE2 correlated with increased intracellular cyclic adenosine monophosphate (cAMP) production, and forskolin, an adenosyl cyclase agonist, confirmed the inhibitory effect of cAMP stimulation on neutrophil phagocytosis. The expression of EP2 receptors by HL‐60 cells was confirmed by western blot analysis, and selective agonism of EP2 receptors mimicked the inhibition of phagocytosis by PGE2. The EP2 receptor antagonist AH‐6089 partially blocked the inhibition of neutrophil phagocytosis PGE2. Specific inhibition of phosphatase and tensin homolog (PTEN) enzyme attenuated the inhibition of neutrophil phagocytosis by PGE2, and both PGE2 and increased intracellular cAMP increased neutrophil PTEN activity, which was associated with decreased PTEN phosphorylation. The results support negative regulation of the antimicrobial activity of neutrophils (i.e., phagocytosis), which has important implications for the future management of bacterial infections.

Lubiprostone is non-selective activator of cAMP-gated ion channels and Clc-2 has a minor role in its prosecretory effect in intestinal epithelial cells.

Loss of prosecretory Cl- channel CFTR activity is considered as the key cause of gastrointestinal disorders in cystic fibrosis including constipation and meconium ileus. Clc-2 is proposed as an alternative Cl- channel in intestinal epithelia that can compensate for CFTR loss-of-function. Lubiprostone is an FDA-approved drug with Clc-2 activation as its presumed mechanism of action. However, relative contribution of Clc-2 in intestinal Cl- secretion and the mechanism of action of lubiprostone remain controversial due to lack of selective Clc-2 inhibitors. Using recently identified selective Clc-2 inhibitor AK-42, we characterized the roles of Clc-2 in Cl- secretion in human intestinal epithelial T84 cells. Clc-2 inhibitor AK-42 had minimal (15%) inhibitory effect on secretory short-circuit current (Isc) induced by cAMP agonists, where subsequently applied CFTR inhibitor (CFTRinh-172) caused 2-3 fold greater inhibition. Similarly, AK-42 inhibited lubiprostone-induced secretory Isc by 20%, whereas CFTRinh-172 caused 2-3 fold greater inhibition. In addition to increasing CFTR and Clc-2-mediated apical Cl- conductance, lubiprostone increased basolateral membrane K+ conductance, which was completely reversed by cAMP-activated K+ channel inhibitor BaCl2 All components of lubiprostone-induced secretion (Clc-2, CFTR and K+ channels) were inhibited by ~65% with the extracellular Ca2+-sensing receptor (CaSR) activator cinacalcet that stimulates cAMP hydrolysis. Lastly, EP4 prostaglandin receptor inhibitor GW627368 pretreatment inhibited lubiprostone-induced secretion by 40% without any effect on forskolin response. Our findings suggest that Clc-2 has minor role in cAMP-induced intestinal Cl- secretion; and lubiprostone is not a selective Clc-2 activator, but general activator of cAMP-gated ion channels in human intestinal epithelial cells. Significance Statement Cl- channel Clc-2 activation is the proposed mechanism of action of the FDA-approved constipation drug lubiprostone. Using first-in-class selective Clc-2 inhibitor AK-42, we showed that Clc-2 has minor contribution in intestinal Cl- secretion induced by lubiprostone and cAMP agonists. We also found that lubiprostone is a general activator of cAMP-gated ion channels in human intestinal epithelial cells (via EP4 receptors). Our findings clarify the roles of Clc-2 in intestinal Cl- secretion and elucidate the mechanism of action of approved-drug lubiprostone.

Identification and Characterization of Human Colorectal Cancer Cluster Predominantly Expressing EP3 Prostanoid Receptor Subtype.

Colorectal cancer (CRC) is one of the common types of cancer in humans. Prostaglandin E2 (PGE2) is a well-known mediator of colorectal cancer through stimulation of four E-type prostanoid (EP) receptor subtypes: EP1, EP2, EP3, and EP4 receptors. All subtypes of EP receptors are involved in CRC promotion or malignancy. However, the characteristics of CRC that highly expresses EP receptor subtypes have not been clarified. In the present study, we classified CRC from a cancer genomic database and identified CRC clusters which highly express EP receptor subtypes. Most of these clusters predominantly expressed one subtype of EP receptor and showed different gene expression patterns. Among them, we focused on the cluster highly expressing the EP3 receptor (CL-EP3). As the result of characterization of gene expression, CL-EP3 was characterized as: epithelial mesenchymal transition (EMT)-induced progressed cancer with activation of transforming growth factor-β pathway, activation of hypoxia-inducible factor-1α, and suppression of runt-related transcription factor 3. Since we previously reported that EP3 receptor is involved in and induce colon cancer cell migration, EP3 receptor-expressing CRC may induce metastasis through these signaling pathways. Thus, the findings suggest the effectiveness of cancer clustering by gene expression of the EP receptor subtype to elucidate the mechanism of human CRC.

A phase 1 study of TPST-1495 as a single agent and in combination with pembrolizumab in subjects with solid tumors.

TPS2696 Background: Prostaglandin E2 (PGE2) is a bioactive lipid that promotes cancer through diverse mechanisms including stimulating tumor proliferation, enhancing angiogenesis and suppressing immune function in the tumor microenvironment. PGE2 produced by tumor cells through upregulation of cyclooxygenase-2 (COX-2) is also a key mediator of adaptive resistance to immune checkpoint inhibitor therapy. While PGE2 signaling is important in cancer, how best to inhibit PGE2 for cancer treatment is under investigation. Inhibition of COX enzymes (e.g., with NSAIDS) has shown promise in large observational studies, but inconsistent results in prospective studies. Importantly, COX inhibition alters multiple prostanoids beyond PGE2, resulting in toxicity that limits therapeutic dosing for cancer. PGE2 signals through four receptors, EP1-4, that are variably expressed and have distinct activities. The tumor promoting and immunosuppressive activities of PGE2 predominantly arise from signaling through the EP2 and EP4 receptors, while signaling through the EP1 and EP3 receptors generally is pro-inflammatory. TPST-1495 is designed to be an oral, highly specific, antagonist of the EP2 and EP4 receptors, sparing the EP1 and EP3 receptors and the COX enzymes. Preclinical studies suggest that blocking EP2 and EP4 with TPST-1495 inhibits tumor proliferation and stimulates anti-cancer immunity better than inhibiting all 4 receptors together, the EP2 or EP4 receptors singly, or the upstream COX-2 enzyme. Methods: TPST-1495-001 is a first-in-human Phase 1 study (NCT04344795). In the Dose and Schedule Optimization Stage, the primary objectives are to characterize the safety and tolerability (including dose limiting toxicities) and determine the recommended phase 2 dose (RP2D) of TPST-1495 as monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. Additional objectives include characterization of PK, PD, and evaluation of potential biomarkers, including through paired (pre- and on-treatment) tumor biopsies. Monotherapy dose-finding employs a modified 3+3 design evaluating BID and QD TPST-1495 schedules along with continuous or intermittent (Days 1-5 every 7 days) dosing. For the pembrolizumab combination, the starting dose and schedule of TPST-1495 are determined by safety, PK and PD of monotherapy. Indication-specific Expansion Stage cohorts will evaluate TPST-1495 at the selected RP2D and schedule for both monotherapy and combination in endometrial cancer, squamous cell carcinoma of the head and neck, and microsatellite stable colorectal cancer (combination only), as well as in a biomarker-specific cohort enrolling patients with pathogenic tumor PIK3CA gene mutation. Dose and Schedule Optimization enrollment (monotherapy and combination) is ongoing at abstract submission while Expansion Stages are planned after identification of the RP2Ds. Clinical trial information: NCT04344795.

AUGMENTED PROSTAGLANDIN E2 SIGNALING CAUSES PODOCYTE DAMAGE AND ALBUMINURIA-LIKE PHENOTYPE IN A ZEBRAFISH DISEASE MODEL OF GLOMERULAR HYPERFILTRATION

Objective: Prostaglandins (PGs) are important active lipids synthesized from arachidonic acid with the participation of cyclooxygenases (COXs) and specific prostaglandin synthases. Prostaglandin E2 (PGE2), the most abundant prostaglandin in the kidney, plays a crucial role in renal homeostasis and the pathogenesis of chronic kidney diseases. Augmented PGE2 signaling that may occur in podocytes during increased fluid flow shear stress as observed during glomerular hyperfiltration, results in glomerular filtration barrier (GFB) permeability defects, ultimately leading to albuminuria. The cellular mechanisms that precede podocyte damage are not fully resolved. Here, we deploy a zebrafish model to study the role of PGE2 signaling in glomerulus and its effects on GFB integrity and function. Design and method: To model the outcome of increased PGE2 signaling in podocytes, zebrafish embryos were exposed to 16,16-dimethyl PGE2 (dmPGE2, 125 μM) or DMSO vehicle after the complete formation of GFB, i.e. from 72 hours post fertilization (hpf) to 96 hpf. To probe the PGE2 pathway, embryos were treated with the selective PGE2 receptor (EP) type 2 (EP2) and EP4 antagonists PF04418948 and ONO-AE3-208 or the COX inhibitor indomethacin. Zebrafish embryos of Tg(fabp10a:gc-EGFP) were used to evaluate the functional role of GFB integrity and albuminuria phenotype, while embryos of Tg(wt1b:EGFP) in combination with angiography using BSA-conjugated AlexaFluor555 allowed glomerular morphological analysis. In vivo stereomicroscopy, confocal laser scanning microscopy, and electron microscopy were used to analyze GFB at cellular as well as ultrastructural level. Statistical analysis was performed using GraphPad software. Results: Stimulation with dmPGE2 causes an albuminuria-like phenotype in zebrafish embryos, mimicking the suggested PGE2 effects during glomerular hyperfiltration in rodent models. The albuminuria-like phenotype could be significantly suppressed by the combined and separate inhibition of the EP2 and EP4 receptors as well as by indomethacin treatment. Although the glomerular ultrastructure appeared unaffected, high-resolution confocal imaging showed impaired podocyte intercalation around the glomerular capillaries after dmPGE2 exposure. Importantly, these specific podocyte defects were restored after blockage of EP2 and EP4. Conclusions: Our results support a role of augmented PGE2/EP2/EP4 signaling in the setting of glomerular hyperfiltration and albuminuria.

Targeting EP2 receptor with multifaceted mechanisms for high-risk neuroblastoma.

SUMMARYProstaglandin E2 (PGE2) promotes tumor cell proliferation, migration, and invasion, fostering an inflammation-enriched microenvironment that facilitates angiogenesis and immune evasion. However, the PGE2 receptor subtype (EP1–EP4) involved in neuroblastoma (NB) growth remains elusive. Herein, we show that the EP2 receptor highly correlates with NB aggressiveness and acts as a predominant Gαs-coupled receptor mediating PGE2-initiated cyclic AMP (cAMP) signaling in NB cells with high-risk factors, including 11q deletion and MYCN amplification. Knockout of EP2 in NB cells blocks the development of xenografts, and its conditional knockdown prevents established tumors from progressing. Pharmacological inhibition of EP2 by our recently developed antagonist TG6-129 suppresses the growth of NB xenografts in nude mice and syngeneic allografts in immunocompetent hosts, accompanied by anti-inflammatory, antiangiogenic, and apoptotic effects. This proof-of-concept study suggests that the PGE2/EP2 signaling pathway contributes to NB malignancy and that EP2 inhibition by our drug-like compounds provides a promising strategy to treat this deadly pediatric cancer.

GLOMERULAR PROSTAGLANDIN E2 IS UPREGULATED DURING ONSET OF ALBUMINURIA THAT IS SUPPRESSED AFTER DUAL BLOCKADE OF ITS EP2 AND EP4 RECEPTORS IN A RAT MODEL OF GLOMERULAR HYPERFILTRA

Objective: Glomerular hyperfiltration (GH) is an important mechanism in the development of albuminuria in hypertension. Recent in vitro data indicated an activation of an autocrine/paracrine cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) pathway resulting in podocyte injury in GH by using fluid flow shear stress exposure. This was mediated by concerted ignalling via both prostaglandin E2 receptors (EP), EP2 and EP4. Here, we explored the role to target this pathway in vivo by using a rat model with GH and albuminuria. Design and method: We used the Munich Wistar Frömter (MWF) rat model with an inherited nephron deficit as a model of GH and albuminuria. Because analysis of PGE2 levels in vivo is limited by conventional methods, we developed a modified liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) protocol to quantify PGE2 levels in glomerular tissue. We then assessed glomerular PGE2 during the onset of albuminuria in young MWF at 4 to 8 weeks of age. Furthermore, the renoprotective effects of either separate or combined pharmacological blockade of EP2 (by PF-04418948) and EP4 (by ONO-AE3-208) in MWF was explored. Treatment started at the onset of albuminuria at 4 weeks and was continued for 8 weeks. Results: During the onset of albuminuria, PGE2 levels were significantly elevated in isolated glomeruli of MWF at both 4 and 8 weeks of age (p &lt; 0.01, respectively) as compared to albuminuria resistant spontaneously hypertensive rats (SHR). At 12 weeks of age, untreated MWF developed pronounced albuminuria, which was significantly, although modestly, reduced by separate EP2 or EP4 blockade. Remarkably, combined EP2 and EP4 blockade resulted in a significant suppression (-71%) of albuminuria in MWF at 12 weeks. Importantly, this pharmacological treatment did not affect systolic blood pressure and creatinine clearance (Figure 1). Conclusions: We demonstrated the activation of glomerular PGE2 in vivo in the setting of hyperfiltration and albuminuria in the MWF rat model. Dual EP2 and EP4 receptor blockade exhibited a marked suppression of albuminuria without affecting systemic blood pressure and glomerular filtration rate. These data support further translational research to explore this therapeutic option for renoprotection.

Cyclooxygenase-Derived Prostaglandin E2 Drives IL-1-Independent Mycobacterium bovis Bacille Calmette-Guérin-Triggered Skin Dendritic Cell Migration to Draining Lymph Node.

Inoculation of Mycobacterium bovis Bacille Calmette-Guérin (BCG) in the skin mobilizes local dendritic cells (DC) to the draining lymph node (dLN) in a process that remains incompletely understood. In this study, a mouse model of BCG skin infection was used to investigate mechanisms of skin DC migration to dLNs. We found enhanced transcription of cyclooxygenase (COX)-2 and production of COX-derived PGE2 early after BCG infection in skin. Animals treated with antagonists for COX or the PGE2 receptors EP2 and EP4 displayed a marked reduction in the entry of skin DCs and BCG to dLNs, uncovering an important contribution of COX-derived PGE2 in this migration process. In addition, live BCG bacilli were needed to invoke DC migration through this COX-PGE2 pathway. Having previously shown that IL-1R partially regulates BCG-induced relocation of skin DCs to dLNs, we investigated whether PGE2 release was under control of IL-1. Interestingly, IL-1R ligands IL-1α/β were not required for early transcription of COX-2 or production of PGE2 in BCG-infected skin, suggesting that the DC migration-promoting role of PGE2 is independent of IL-1α/β in our model. In DC adoptive transfer experiments, EP2/EP4, but not IL-1R, was needed on the moving DCs for full-fledged migration, supporting different modes of action for PGE2 and IL-1α/β. In summary, our data highlight an important role for PGE2 in guiding DCs to dLNs in an IL-1–independent manner.

Pharmacokinetics of grapiprant and effects on TNF-alpha concentrations following oral administration to horses.

Grapiprant is a prostaglandin E2 receptor antagonist that has been found to be an effective anti-inflammatory in dogs and that is devoid of some of the adverse effects associated with traditional NSAIDs that elicit their effects through inhibition of PGE2 production. Previously published reports have described the pharmacokinetics of this drug in horses when administered at 2 mg/kg; however, pharmacodynamic effects in this species have yet to be described. The objective of the current study was to describe the pharmacokinetics and pharmacodynamics of grapiprant at a higher dose. Eight horses received a single oral administration of 15 mg/kg. Plasma concentrations were determined for 96 h using liquid chromatography-tandem mass spectrometry. Non-compartmental analysis was used to determine pharmacokinetic parameters. Pharmacodynamic effects were assessed ex vivo by stimulating blood samples with PGE2 and determining TNF-ɑ concentrations. Maximum concentration, time to maximum concentration and area under the curve were 327.5 (188.4-663.0) ng/ml, 1 (0.75-2.0) hour and 831.8 (512.6-1421.6) h*ng/ml, respectively. The terminal half-life was 11.1 (8.27-21.2) hr. Significant stimulation of TNF alpha was noted for 2-4h post-drug administration. Results of this study suggest a short duration of EP4 receptor engagement when administered at a dose of 15 mg/kg.

Scaffold Hopping Strategy to Identify Prostanoid EP4 Receptor Antagonists for Cancer Immunotherapy.

Cancer cells can effectively suppress the natural immune response in humans, and prostaglandin E2 (PGE2) is a key mediator in the development of tumor cell resistance to immunotherapy. As a major contributor to PGE2-elicited immunosuppressive activity, the EP4 receptor promotes tumor development and progression in the tumor microenvironment, and the development of selective and potent EP4 receptor antagonists should have promising potential for tumor immunotherapy. Aiming at improving the drug-like properties, a series of 4,7-dihydro-5H-thieno[2,3-c]pyran derivatives were designed and synthesized through a scaffold hopping strategy. The most promising compound 47 exhibited good EP4 antagonistic activity and excellent subtype selectivity, as well as favorable drug-like properties. It effectively suppressed the expression of multiple immunosuppression-related genes in macrophages. Meanwhile, oral administration of compound 47, alone or in combination with anti-PD-1 antibody, significantly enhanced the antitumor immune response and inhibited tumor growth in the mouse CT26 colon carcinoma model.