Discovery of the Novel, Orally Active, and Selective LPA1 Receptor Antagonist ACT-1016-0707 as a Preclinical Candidate for the Treatment of Fibrotic Diseases.

Piperidine 3 is a potent and selective lysophosphatidic acid receptor subtype 1 receptor (LPAR1) antagonist that has shown efficacy in a skin vascular leakage target engagement model in mice. However, compound 3 has very high human plasma protein binding and high clearance in rats, which could significantly hamper its clinical development. Continued lead optimization led to the potent, less protein bound, metabolically stable, and orally active azetidine 17. Rat pharmacokinetics (PK) studies revealed that 17 accumulated in the liver. In vitro studies indicated that 17 is an organic anion co-transporting polypeptide 1B1 (OATP1B1) substrate. Although analogue 24 was no longer a substrate of OATP1B1, PK studies suggested that the compound undergoes enterohepatic recirculation. Replacing the carboxylic acidic side chain by a non-acidic sulfamide moiety and further fine-tuning of the scaffold yielded the potent, orally active LPAR1 antagonist 49, which was selected for preclinical development for the treatment of fibrotic diseases.

Global Proteomics Analysis of Lysophosphatidic Acid Signaling in PC-3 Human Prostate Cancer Cells: Role of CCN1.

Cysteine-rich angiogenic factor 61 (CCN1/Cyr61) is a matricellular protein that is induced and secreted in response to growth factors. Our previous work showed that 18:1-lysophosphatidic acid (LPA), which activates the G protein-coupled receptor LPAR1, induces CCN1 between 2-4 h in PC-3 human prostate cancer cells in a manner than enhances cell-substrate adhesion. While the time course of induction suggests that CCN1 contributes to intermediate events in LPA action, the roles of CCN1 in LPA-mediated signal transduction have not been fully elucidated. This study utilized a comprehensive global proteomics approach to identify proteins up- or down-regulated in response to treatment of PC-3 cells with LPA for three hours, during the time of peak CCN1 levels. In addition, the effects of siRNA-mediated CCN1 knockdown on LPA responses were analyzed. The results show that, in addition to CCN1, LPA increased the levels of multiple proteins. Proteins up-regulated by LPA included metastasis-associated in colon cancer protein 1 (MACC1) and thrombospondin-1 (TSP1/THBS1); both MACC1 and TSP1 regulated cancer cell adhesion and motility. LPA down-regulated thioredoxin interacting protein (TXNIP). CCN1 knockdown suppressed the LPA-induced up-regulation of 30 proteins; these included MACC1 and TSP1, as confirmed by immunoblotting. Gene ontology and STRING analyses revealed multiple pathways impacted by LPA and CCN1. These results indicate that CCN1 contributes to LPA signaling cascades that occur during the intermediate phase after the initial stimulus. The study provides a rationale for the development of interventions to disrupt the LPA-CCN1 axis.

Network analysis-guided drug repurposing strategies targeting LPAR receptor in the interplay of COVID, Alzheimer's, and diabetes.

The COVID-19 pandemic caused by the SARS-CoV-2 virus has greatly affected global health. Emerging evidence suggests a complex interplay between Alzheimer's disease (AD), diabetes (DM), and COVID-19. Given COVID-19's involvement in the increased risk of other diseases, there is an urgent need to identify novel targets and drugs to combat these interconnected health challenges. Lysophosphatidic acid receptors (LPARs), belonging to the G protein-coupled receptor family, have been implicated in various pathological conditions, including inflammation. In this regard, the study aimed to investigate the involvement of LPARs (specifically LPAR1, 3, 6) in the tri-directional relationship between AD, DM, and COVID-19 through network analysis, as well as explore the therapeutic potential of selected anti-AD, anti-DM drugs as LPAR, SPIKE antagonists. We used the Coremine Medical database to identify genes related to DM, AD, and COVID-19. Furthermore, STRING analysis was used to identify the interacting partners of LPAR1, LPAR3, and LPAR6. Additionally, a literature search revealed 78 drugs on the market or in clinical studies that were used for treating either AD or DM. We carried out docking analysis of these drugs against the LPAR1, LPAR3, and LPAR6. Furthermore, we modeled the LPAR1, LPAR3, and LPAR6 in a complex with the COVID-19 spike protein and performed a docking study of selected drugs with the LPAR-Spike complex. The analysis revealed 177 common genes implicated in AD, DM, and COVID-19. Protein-protein docking analysis demonstrated that LPAR (1,3 & 6) efficiently binds with the viral SPIKE protein, suggesting them as targets for viral infection. Furthermore, docking analysis of the anti-AD and anti-DM drugs against LPARs, SPIKE protein, and the LPARs-SPIKE complex revealed promising candidates, including lupron, neflamapimod, and nilotinib, stating the importance of drug repurposing in the drug discovery process. These drugs exhibited the ability to bind and inhibit the LPAR receptor activity and the SPIKE protein and interfere with LPAR-SPIKE protein interaction. Through a combined network and targeted-based therapeutic intervention approach, this study has identified several drugs that could be repurposed for treating COVID-19 due to their expected interference with LPAR(1, 3, and 6) and spike protein complexes. In addition, it can also be hypothesized that the co-administration of these identified drugs during COVID-19 infection may not only help mitigate the impact of the virus but also potentially contribute to the prevention or management of post-COVID complications related to AD and DM.

Roles of lysophosphatidic acid (LPA) receptor-mediated signaling in cellular functions modulated by endothelial cells in pancreatic cancer cells under hypoxic conditions.

BACKGROUND: In the tumor environment, malignant characteristics of cancer cells are promoted by stromal cells under hypoxia. It is unknown whether lysophosphatidic acid (LPA) receptor-mediated signaling is involved in the regulation of cellular functions by endothelial cells in pancreatic cancer cells under hypoxic conditions. METHODS: Pancreatic cancer (PANC-1) cells were co-cultured with endothelial (F2) cells and F2 cell supernatants at 21% and 1% O2. The Cell Culture Insert was used to assess the cell motile activity. The cell growth and viability to cisplatin (CDDP) were measured, using the Cell Counting Kit-8. RESULTS: LPA receptor expression levels were changed in PANC-1 cells co-cultured with F2 cells at 21% and 1% O2. The cell motile activities of PANC-1 cells co-cultured with F2 cells at 21% and 1% O2 were markedly elevated, compared with PANC-1 cells alone. The cell viabilities to CDDP of PANC-1 cells co-cultured with F2 cell supernatants at 21% and 1% O2 were regulated by the activation of LPA receptors. CONCLUSION: These results suggest that LPA receptor-mediated signaling plays an important role in the modulation of pancreatic cancer cell functions by endothelial cells under hypoxic conditions.

Lysophosphatidic Acid Receptor 3 Activation Is Involved in the Regulation of Ferroptosis.

Ferroptosis, a unique form of programmed cell death trigged by lipid peroxidation and iron accumulation, has been implicated in embryonic erythropoiesis and aging. Our previous research demonstrated that lysophosphatidic acid receptor 3 (LPA3) activation mitigated oxidative stress in progeria cells and accelerated the recovery of acute anemia in mice. Given that both processes involve iron metabolism, we hypothesized that LPA3 activation might mediate cellular ferroptosis. In this study, we used an LPA3 agonist, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate (OMPT), to activate LPA3 and examine its effects on the ferroptosis process. OMPT treatment elevated anti-ferroptosis gene protein expression, including solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and ferritin heavy chain (FTH1), in erastin-induced cells. Furthermore, OMPT reduced lipid peroxidation and intracellular ferrous iron accumulation, as evidenced by C11 BODIPY™ 581/591 Lipid Peroxidation Sensor and FerroOrange staining. These observations were validated by applying LPAR3 siRNA in the experiments mentioned above. In addition, the protein expression level of nuclear factor erythroid 2-related factor (NRF2), a key regulator of oxidative stress, was also enhanced in OMPT-treated cells. Lastly, we verified that LPA3 plays a critical role in erastin-induced ferroptotic human erythroleukemia K562 cells. OMPT rescued the erythropoiesis defect caused by erastin in K562 cells based on a Gly A promoter luciferase assay. Taken together, our findings suggest that LPA3 activation inhibits cell ferroptosis by suppressing lipid oxidation and iron accumulation, indicating that ferroptosis could potentially serve as a link among LPA3, erythropoiesis, and aging.

Phosphatidylethanolamine (18:2e/18:2) may inhibit adipose tissue wasting in patients with cancer cachexia by increasing lysophosphatidic acid receptor 6.

BACKGROUND: Cancer associated cachexia is characterized by the significant loss of adipose tissue, leading to devastating weight loss and muscle wasting in the majority of cancer patients. The effects and underlying mechanisms of degradation metabolites on adipocytes in cachectic patients remain poorly understood. To address this knowledge gap, we conducted a comprehensive study combining lipidomic analysis of subcutaneous and visceral adipose tissue with transcriptomics data from the database to investigate the mechanisms of lipid regulation in adipocytes. METHODS: We collected subcutaneous and visceral adipose tissue samples from cachectic and noncachectic cancer patients. Lipidomic analysis was performed to identify differentially expressed lipids in both types of adipose tissue. Additionally, transcriptomics data from the GEO database were analyzed to explore gene expression patterns in adipocytes. Bioinformatics analysis was employed to determine the enrichment of differentially expressed genes in specific pathways. Furthermore, molecular docking studies were conducted to predict potential protein targets of specific lipids, with a focus on the PI3K-Akt signaling pathway. Western blot analysis was used to validate protein levels of the identified target gene, lysophosphatidic acid receptor 6 (LPAR6), in subcutaneous and visceral adipose tissue from cachectic and noncachectic patients. RESULTS: Significant lipid differences in subcutaneous and visceral adipose tissue between cachectic and noncachectic patients were identified by multivariate statistical analysis. Cachectic patients exhibited elevated Ceramides levels and reduced CerG2GNAc1 levels (P < 0.05). A total of 10 shared lipids correlated with weight loss and IL-6 levels, enriched in Sphingolipid metabolism, GPI-anchor biosynthesis, and Glyceropholipid metabolism pathways. LPAR6 expression was significantly elevated in both adipose tissues of cachectic patients (P < 0.05). Molecular docking analysis indicated strong binding of Phosphatidylethanolamine (PE) (18:2e/18:2) to LPAR6. CONCLUSIONS: Our findings suggest that specific lipids, including PE(18:2e/18:2), may mitigate adipose tissue wasting in cachexia by modulating the expression of LPAR6 through the PI3K-Akt signaling pathway. The identification of these potential targets and mechanisms provides a foundation for future investigations and therapeutic strategies to combat cachexia. By understanding the underlying lipid regulation in adipocytes, we aim to develop targeted interventions to ameliorate the devastating impact of cachexia on patient outcomes and quality of life. Nevertheless, further studies and validation are warranted to fully elucidate the intricate mechanisms involved and translate these findings into effective clinical interventions.

Altered ovarian tissue level of lysophosphatidic acid and mRNA expressions of its metabolic enzymes and receptors in rats received gonadotropin-hyperstimulation.

Lysophosphatidic acid (LPA), a well-studied member of the lysophospholipid family, is known to exert an important bio-effect on oocyte maturation and ovulation in mammals. We attempted to determine how follicle maturation in the rat ovary affects the levels of LPA and its precursor lysophospholipids, as well as mRNA levels of LPA-producing and -degrading enzymes and LPA receptors in rats that received gonadotropin-hyper-stimulation. Tissue levels of lysophospholipids were quantified by LC-MS/MS, and relative mRNA expression levels of LPA-producing and -degrading enzymes, and LPA receptors were measured by RT-PCR. Tissue levels of n-6 polyunsaturated LPAs and LPCs were higher in the ovaries of rats after receiving human chorionic gonadotropin, unlike the distinct profiles of n-3 polyunsaturated LPAs, which had lower levels, and LPCs which had higher levels, after the gonadotropin treatment. The effects of different levels of other polyunsaturated lysophospholipids were variable: decreased levels of lysophosphatidylglycerol, and unaltered levels of lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine. The results indicate that expression of mRNA levels of autotaxin and acylglycerol kinase were reduced and expression of lipid phosphate phosphatase 3 was elevated, whereas expressions of two membrane phosphatidic acid phosphatases (A1α and A1ß) and lipid phosphate phosphatase 1 were essentially unaltered in rat ovary at several stages after ovary hyperstimulation. After the gonadotropin treatment, the expression levels of all LPA receptors except LPA3 were decreased at various times. These results are discussed with respect to the physiological processes of the ovarian environment and development in rats.

A dual role of lysophosphatidic acid type 2 receptor (LPAR2) in nonsteroidal anti-inflammatory drug-induced mouse enteropathy.

Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator that has been found to ameliorate nonsteroidal anti-inflammatory drug (NSAID)-induced gastric injury by acting on lysophosphatidic acid type 2 receptor (LPAR2). In this study, we investigated whether LPAR2 signaling was implicated in the development of NSAID-induced small intestinal injury (enteropathy), another major complication of NSAID use. Wild-type (WT) and Lpar2 deficient (Lpar2-/-) mice were treated with a single, large dose (20 or 30 mg/kg, i.g.) of indomethacin (IND). The mice were euthanized at 6 or 24 h after IND treatment. We showed that IND-induced mucosal enteropathy and neutrophil recruitment occurred much earlier (at 6 h after IND treatment) in Lpar2-/- mice compared to WT mice, but the tissue levels of inflammatory mediators (IL-1ß, TNF-α, inducible COX-2, CAMP) remained at much lower levels. Administration of a selective LPAR2 agonist DBIBB (1, 10 mg/kg, i.g., twice at 24 h and 30 min before IND treatment) dose-dependently reduced mucosal injury and neutrophil activation in enteropathy, but it also enhanced IND-induced elevation of several proinflammatory chemokines and cytokines. By assessing caspase-3 activation, we found significantly increased intestinal apoptosis in IND-treated Lpar2-/- mice, but it was attenuated after DBIBB administration, especially in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Finally, we showed that IND treatment reduced the plasma activity and expression of autotaxin (ATX), the main LPA-producing enzyme, and also reduced the intestinal expression of Lpar2 mRNA, which preceded the development of mucosal damage. We conclude that LPAR2 has a dual role in NSAID enteropathy, as it contributes to the maintenance of mucosal integrity after NSAID exposure, but also orchestrates the inflammatory responses associated with ulceration. Our study suggests that IND-induced inhibition of the ATX-LPAR2 axis is an early event in the pathogenesis of enteropathy.

Disruption of LPA-LPAR1 pathway results in lung tumor growth inhibition by downregulating B7-H3 expression in fibroblasts.

BACKGROUND: Lysophosphatidic acids (LPAs) belong to a class of bioactive lysophospholipids with multiple functions including immunomodulatory roles in tumor microenvironment (TME). LPA exerts its biological effects via its receptors that are highly expressed in fibroblasts among other cell types. As cancer-associated fibroblasts (CAFs) are a key component of the TME, it is important to understand LPA signaling and regulation of receptors in fibroblasts or CAFs and associated regulatory roles on immunomodulation-related molecules. METHODS: Cluster analysis, immunoblotting, real-time quantitative-PCR, CRISPR-Cas9 gene editing system, immunohistochemical staining, coculture model, and in vivo xenograft model were used to investigate the effects of LPA-LPAR1 on B7-H3 in tumor promotion of CAFs. RESULTS: In this study, we found that LPAR1 and CD276 (B7-H3) were generally highly expressed in fibroblasts with good expression correlation. LPA induced B7-H3 up-expression through LPAR1, and stimulated fibroblasts proliferation that could be inhibited by silencing LPAR1 or B7-H3 as well as small molecule LPAR1 antagonist (Ki16425). Using engineered fibroblasts and non-small cell lung carcinoma (NSCLC) cell lines, subsequent investigations demonstrated that CAFs promoted the proliferation of NSCLC in vitro and in vivo, and such effect could be inhibited by knocking out LPAR1 or B7-H3. CONCLUSION: The present study provided new insights for roles of LPA in CAFs, which could lead to the development of innovative therapies targeting CAFs in the TME. It is also reasonable to postulate a combinatory approach to treat malignant fibrous tumors (such as NSCLC) with LPAR1 antagonists and B7-H3 targeting therapies.

Integration of an LPAR1 Antagonist into Liposomes Enhances Their Internalization and Tumor Accumulation in an Animal Model of Human Metastatic Breast Cancer.

Lysophosphatidic acid receptor 1 (LPAR1) is elevated in breast cancer. The deregulation of LPAR1, including the function and level of expression, is linked to cancer initiation, progression, and metastasis. LPAR1 antagonists, AM095 or Ki16425, may be effective therapeutic molecules, yet their limited water solubility hinders in vivo delivery. In this study, we report on the synthesis of two liposomal formulations incorporating AM095 or Ki16425, embedded within the lipid bilayer, as targeted nanocarriers for metastatic breast cancer (MBC). The data show that the Ki16425 liposomal formulation exhibited a 50% increase in internalization by MBC mouse epithelial cells (4T1) and a 100% increase in tumor accumulation in a mouse model of MBC compared with that of a blank liposomal formulation (control). At the same time, normal mouse epithelial cells (EpH-4Ev) internalized the Ki16425 liposomal formulation 25% lesser than the control formulation. Molecular dynamics simulations show that the integration of AM095 or Ki16425 modified the physical and mechanical properties of the lipid bilayer, making it more flexible in these liposomal formulations compared with liposomes without drug. The incorporation of an LPAR1 antagonist within a liposomal drug delivery system represents a viable therapeutic approach for targeting the LPA-LPAR1 axis, which may hinder the progression of MBC.

Expression of lysophosphatidic acid receptors in the rat uterus: cellular distribution of protein and gestation-associated changes in gene expression.

Though lysophosphatidic acid (LPA) shows a variety of regulatory roles in reproduction, its action mechanisms in the gestational organs are still largely unknown. We here characterized cellular distribution of its six kinds of specific receptors (LPA1-6) in rat uteri by immunohistochemistry and quantitatively analyzed changes in Lpar1-6 mRNAs expression throughout pregnancy. Among LPA1-6, evident expression of LPA3, LPA4, and LPA6 was immunologically detected and less expression of immunoreactive LPA1 and LPA2 was also found. Luminal and glandular epithelial cells, stromal cells, and myometrial cells are sites of positive immunoreactions, and they are all likely to express three or more subtypes. All of Lpar1-6 mRNAs were expressed, and their alterations were variable depending on subtypes and gestational age. The present information suggests that diverse actions of LPA in the uterus involve varied expression of LPA receptors dependent on tissue/cell types, receptor subtype(s), and organ reproductive states and helps to understand uterine biology of LPA.

Autotaxin-lysophosphatidic acid receptor 5 axis evokes endothelial dysfunction via reactive oxygen species signaling.

Lysophosphatidylcholine (LPC) is a bioactive lipid that has been shown to attenuate endothelium-dependent vasorelaxation contributing to endothelial dysfunction; however, the underlying mechanisms are not well understood. In this study, we investigated the molecular mechanisms involved in the development of LPC-evoked impairment of endothelium-dependent vasorelaxation. In aortic rings isolated from wild-type (WT) mice, a 20-min exposure to LPC significantly reduced the acetylcholine chloride (ACh)-induced vasorelaxation indicating the impairment of normal endothelial function. Interestingly, pharmacological inhibition of autotaxin (ATX) by GLPG1690 partially reversed the endothelial dysfunction, suggesting that lysophosphatidic acid (LPA) derived from LPC may be involved in the effect. Therefore, the effect of LPC was also tested in aortic rings isolated from different LPA receptor knock-out (KO) mice. LPC evoked a marked reduction in ACh-dependent vasorelaxation in Lpar1, Lpar2, and Lpar4 KO, but its effect was significantly attenuated in Lpar5 KO vessels. Furthermore, addition of superoxide dismutase reduced the LPC-induced endothelial dysfunction in WT but not in the Lpar5 KO mice. In addition, LPC increased H2O2 release from WT vessels, which was significantly reduced in Lpar5 KO vessels. Our findings indicate that the ATX-LPA-LPA5 receptor axis is involved in the development of LPC-induced impairment of endothelium-dependent vasorelaxation via LPA5 receptor-mediated reactive oxygen species production. Taken together, in this study, we identified a new pathway contributing to the development of LPC-induced endothelial dysfunction.

Differential targeting of lysophosphatidic acid LPA1, LPA2, and LPA3 receptor signalling by tricyclic and tetracyclic antidepressants.

We previously reported that in different cell types antidepressant drugs activate lysophosphatidic acid (LPA) LPA1 receptor to induce proliferative and prosurvival responses. Here, we further characterize this unique action of antidepressants by examining their effects on two additional LPA receptor family members, LPA2 and LPA3. Human LPA1-3 receptors were stably expressed in HEK-293 cells (HEK-LPA1, -LPA2 and -LPA3 cells) and their functional activity was determined by Western blot and immunofluorescence. LPA effectively stimulated the phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in HEK-LPA1, -LPA2, and -LPA3 cells. The tricyclic antidepressants amitriptyline, clomipramine, imipramine and desipramine increased phospho-ERK1/2 levels in HEK-LPA1 and -LPA3 cells but were relatively poor agonists in LPA2-expressing cells. The tetracyclic antidepressants mianserin and mirtazapine were active at all three LPA receptors. When combined with LPA, both amitriptyline and mianserin potentiated Gi/o-mediated phosphorylation of ERK1/2 induced by LPA in HEK-LPA1, -LPA2 and -LPA3 cells, CHO-K1 fibroblasts and HT22 hippocampal neuroblasts. This potentiation was associated with enhanced phosphorylation of CREB and S6 ribosomal protein, two molecular targets of activated ERK1/2. The antidepressants also potentiated LPA-induced Gq/11-mediated phosphorylation of AMP-activated protein kinase in HEK-LPA1 and -LPA3 cells. Conversely, amitriptyline and mianserin were found to inhibit LPA-induced Rho activation in HEK-LPA1 and LPA2 cells. These results indicate that tricyclic and tetracyclic antidepressants can act on LPA1, LPA2 and LPA3 receptor subtypes and exert differential effects on LPA signalling through these receptors.

PARP1 modulates METTL3 promoter chromatin accessibility and associated LPAR5 RNA m6A methylation to control cancer cell radiosensitivity.

Chromatin remodeling and N6-methyladenosine (m6A) modification are two critical layers in controlling gene expression and DNA damage signaling in most eukaryotic bioprocesses. Here, we report that poly(ADP-ribose) polymerase 1 (PARP1) controls the chromatin accessibility of METTL3 to regulate its transcription and subsequent m6A methylation of poly(A)+ RNA in response to DNA damage induced by radiation. The transcription factors nuclear factor I-C (NFIC) and TATA binding protein (TBP) are dependent on PARP1 to access the METTL3 promoter to activate METTL3 transcription. Upon irradiation or PARP1 inhibitor treatment, PARP1 disassociated from METTL3 promoter chromatin, which resulted in attenuated accessibility of NFIC and TBP and, consequently, suppressed METTL3 expression and RNA m6A methylation. Lysophosphatidic Acid Receptor 5 (LPAR5) mRNA was identified as a target of METTL3, and m6A methylation was located at A1881. The level of m6A methylation of LPAR5 significantly decreased, along with METTL3 depression, in cells after irradiation or PARP1 inhibition. Mutation of the LPAR5 A1881 locus in its 3' UTR results in loss of m6A methylation and, consequently, decreased stability of LPAR5 mRNA. METTL3-targeted small-molecule inhibitors depress murine xenograft tumor growth and exhibit a synergistic effect with radiotherapy in vivo. These findings advance our comprehensive understanding of PARP-related biological roles, which may have implications for developing valuable therapeutic strategies for PARP1 inhibitors in oncology.

Lysophosphatidic Acid Receptor Signaling in the Human Breast Cancer Tumor Microenvironment Elicits Receptor-Dependent Effects on Tumor Progression.

Lysophosphatidic acid receptors (LPARs) are six G-protein-coupled receptors that mediate LPA signaling to promote tumorigenesis and therapy resistance in many cancer subtypes, including breast cancer. Individual-receptor-targeted monotherapies are under investigation, but receptor agonism or antagonism effects within the tumor microenvironment following treatment are minimally understood. In this study, we used three large, independent breast cancer patient cohorts (TCGA, METABRIC, and GSE96058) and single-cell RNA-sequencing data to show that increased tumor LPAR1, LPAR4, and LPAR6 expression correlated with a less aggressive phenotype, while high LPAR2 expression was particularly associated with increased tumor grade and mutational burden and decreased survival. Through gene set enrichment analysis, it was determined that cell cycling pathways were enriched in tumors with low LPAR1, LPAR4, and LPAR6 expression and high LPAR2 expression. LPAR levels were lower in tumors over normal breast tissue for LPAR1, LPAR3, LPAR4, and LPAR6, while the opposite was observed for LPAR2 and LPAR5. LPAR1 and LPAR4 were highest in cancer-associated fibroblasts, while LPAR6 was highest in endothelial cells, and LPAR2 was highest in cancer epithelial cells. Tumors high in LPAR5 and LPAR6 had the highest cytolytic activity scores, indicating decreased immune system evasion. Overall, our findings suggest that potential compensatory signaling via competing receptors must be considered in LPAR inhibitor therapy.

Lysophosphatidic acid modulates CD8 T cell immunosurveillance and metabolism to impair anti-tumor immunity.

Lysophosphatidic acid (LPA) is a bioactive lipid which increases in concentration locally and systemically across different cancer types. Yet, the exact mechanism(s) of how LPA affects CD8 T cell immunosurveillance during tumor progression remain unknown. We show LPA receptor (LPAR) signaling by CD8 T cells promotes tolerogenic states via metabolic reprogramming and potentiating exhaustive-like differentiation to modulate anti-tumor immunity. We found LPA levels predict response to immunotherapy and Lpar5 signaling promotes cellular states associated with exhausted phenotypes on CD8 T cells. Importantly, we show that Lpar5 regulates CD8 T cell respiration, proton leak, and reactive oxygen species. Together, our findings reveal that LPA serves as a lipid-regulated immune checkpoint by modulating metabolic efficiency through LPAR5 signaling on CD8 T cells. Our study offers key insights into the mechanisms governing adaptive anti-tumor immunity and demonstrates LPA could be exploited as a T cell directed therapy to improve dysfunctional anti-tumor immunity.

Inhibition of lysophosphatidic acid receptor 1 relieves PMN recruitment in CNS via LPA1/TSP1/CXCR2 pathway and alleviates disruption on blood-brain barrier following intracerebral haemorrhage in mice.

BACKGROUD: The frequencies of morbidity and impairment associated with spontaneous intracerebral haemorrhage (ICH) are comparatively high. Blood-brain barrier (BBB) integrity was compromised due to subsequent brain injury induced by ICH, which is crucial for a poor prognosis. Polymorphonuclear leukocyte (PMN) strongly modulate the disruption of BBB in the central nervous system (CNS). The lysophosphatidic acid receptor 1 (LPA1) mediated thrombospondin-1 (TSP1) regulation in astrocytes, which induce macrophage inflammatory protein 2(MIP2) secretion. MIP2 enhance PMN recruitment through CXC chemokine type 2 (CXCR2) activation. The purpose of this study was to investigate whether the LPA1-mediated inhibition of PMN recruitment and BBB protection after ICH is regulated by TSP1 and CXCR2 networks. METHODS: ICH induction was performed in CD1 mice using collagenase administration. AM966, a targeted LPA1 antagonist, was orally administered 1 and 12 h following ICH. further identify possible LPA1-mediated BBB protection mechanisms, we intracerebroventricularly (ICV) administered a CXCR2 ligand MIP2, as well as TSP1 CRISPR activation (ACT) with AM966. Consequently, we performed neurobehavioral, brain water content (BWC), Evans blue staining (EBS), immunofluorescence (IF), and western blot (WB) analyses. RESULTS: After ICH, astrocytes showed signs of LPA1, which peaked after 24 h, while PMN\ displayed evidence of CXCR2. The AM966-mediated LPA1 suppression relieved PMN recruitment, diminished brain oedema, demonstrated extravasation (as evidenced by EBS), protected BBB integrity, and enhanced neurologic activity following ICH. AM966 treatment strongly reduced TSP1, CXCR2, Occludin, and Claudin-5 expressions and PMN recruitment following ICH, and their expressions were restored by MIP2 and TSP1 CRISPR (ACT). CONCLUSIONS: This study shows that LAP1 suppression reduced PMN recruitment after ICH in mice via TSP1/CXCR2 signalling, which minimized BBB disruption and improved the CNS's neurobehavioral functioning. Hence, LPA1 is a strong candidate for therapy to reduce PMN recruitment and offer protection of BBB integrity after ICH.

Combined inhibition of EZH2 and the autotaxin-LPA-LPA2 axis exerts synergistic antitumor effects on colon cancer cells.

Autotaxin (ATX), also known as ENPP2, is the key enzyme in lysophosphatidic acid (LPA) production. LPA acts on its receptors on the cell membrane to promote cell proliferation and migration, and thus, the ATX-LPA axis plays a critical role in tumorigenesis. Clinical data analysis indicated that in colon cancer, there is a strong negative correlation between the expression of ATX and EZH2, the enzymatic catalytic subunit of polycomb repressive complex 2 (PRC2). Here, we demonstrated that ATX expression was epigenetically silenced by PRC2, which was recruited by MTF2 and catalyzed H3K27me3 modification in the ATX promoter region. EZH2 inhibition is a promising strategy for cancer treatment, and ATX expression is induced in colon cancer cells by EZH2 inhibitors. With both EZH2 and ATX as targets, their combined inhibition exerted synergistic antitumor effects on colon cancer cells. In addition, LPA receptor 2 (LPA2) deficiency significantly enhanced the sensitivity to EZH2 inhibitors in colon cancer cells. In summary, our study identified ATX as a novel PRC2 target gene and found that cotargeting EZH2 and the ATX-LPA-LPA2 axis may be a potential combination therapy strategy for colon cancer.

Crosstalk between cannabinoid receptor 2 and lysophosphatidic acid receptor 5.

Cannabinoid receptor 2 (CB2) and lysophosphatidic acid receptor 5 (LPA5) are both classified as G-protein coupled receptors (GPCRs) activated by bioactive lipids and are highly expressed in colon cancer cells. However, crosstalk between two receptors and its potential effects on cancer cell physiology have not been fully elucidated. In the present study, the results of bioluminescence resonance energy transfer analysis showed that, among the LPA receptors, CB2 strongly and specifically interacted with LPA5. Both receptors were co-localized in the plasma membrane in the absence of agonists, and the receptors were co-internalized upon activation of either receptor alone or both receptors together. We further investigated the effects of expression of both receptors on cell proliferation and migration, and the molecular mechanisms underlying these effects in HCT116 colon cancer cells. Co-expression of receptors significantly increased cell proliferation and migration by increasing Akt phosphorylation and tumor progression-related gene expression, whereas no such effect was seen upon expression of either receptor alone. These results suggest the possibility of physical and functional crosstalk between CB2 and LPA5.

Effects of LPA receptor-mediated signaling on the modulation of cellular functions of pancreatic cancer cells cultured in fibroblast supernatants under hypoxic conditions.

The tumor microenvironment (TME) consists of various cell types, including fibroblasts. The TME plays a central role in the promotion of tumor progression. In the present study, we investigated whether lysophosphatidic acid (LPA) receptor-mediated signaling regulates cellular functions by the TME of pancreatic cancer PANC-1 cells. To obtain fibroblast 3T3 cell supernatants, 3T3 cells were cultured in 5% charcoal stripped FCS-DMEM for 48 h. LPAR2 and LPAR3 expression levels were elevated in PANC-1 cells cultured in 3T3 cell supernatants. While PANC-1 cell motility was decreased by 3T3 cell supernatants, the cell survival to cisplatin (CDDP) of PANC-1 cells was markedly enhanced. Moreover, the cell survival to CDDP of PANC-1 cells cultured in 3T3 cell supernatants was increased by GRI-977,143 (LPA2 agonist) and (2 S)-OMPT (LPA3 agonist). Since hypoxia is caused by the restriction of adequate vascular networks to deliver oxygen into solid tumors, PANC-1 cells were cultured in 3T3 cell supernatants at 1% O2 conditions. The cell survival to CDDP of PANC-1 cells cultured in 3T3 cell supernatants at 1% O2 was significantly elevated, correlating with LPAR2 and LPAR3 expressions. These results suggest that LPA signaling via LPA2 and LPA3 is involved in the promotion of malignant properties by the TME in PANC-1 cells.