Ovarian cancer (OC) remains the most lethal gynecologic malignancy, and platinum-taxane combination therapy is the global first-line standard of care for advanced disease. However, resistance to platinum-taxane combination therapy is a major clinical barrier in ovarian cancer (OC), yet the molecular basis of dual resistance remains unresolved. Therefore, defining the molecular mechanisms that drive resistance to combined platinum-taxane therapy is essential for identifying new therapeutic targets and improving patient outcomes. We generated matched RNA-seq and ATAC-seq profiles from parental A2780 cells and derivatives exhibiting functional resistance to platinum (CpR), taxane (TxR), or both agents (TxCpR). We integrated differential gene expression with chromatin accessibility, annotated regulatory elements using ENCODE cCREs, and performed motif enrichment to infer candidate transcription factor programs. We found that CpR and TxR cells displayed the expected drug-specific transcriptional profiles. CpR cells upregulated DNA repair genes, whereas TxR cells showed activation of pathways associated with paclitaxel adaptation, including cytoskeletal regulation and ABCB1-mediated efflux. In contrast, TxCpR cells adopted a distinct regulatory state, separate from CpR and TxR. They exhibited broad enhancer remodeling and enriched accessibility at developmental and morphogenesis-associated loci. Motif analysis identified stress-responsive (MAFF (MAF bzip transcription factor F), NFATC4 (Nuclear factor of activated T-cells 4)) and architectural (YY1 (Yin-Yang 1), ZNF549 (Zinc finger protein 549), CTCF (CCCTC-binding Factor)) transcription factors as candidate regulators of this state. Integrated analysis of RNA-seq and ATAC-seq identified TxCpR-associated candidate distal regulatory elements, including an accessible CTCF motif-containing region near AIM2 (Absent in Melanoma 2) gene, highlighting AIM2-proximal regions as candidates for further functional investigation. Dual platinum-taxane resistance is accompanied by integrated transcriptional plasticity and chromatin rewiring, consistent with a regulatory state that is distinct from single-agent resistance. To our knowledge, this dataset represents the first paired transcriptomic–epigenomic map of all four A2780 states, and identifies candidate transcriptional and distal regulatory elements that may be exploited to target multidrug resistance in OC.

The autotaxin–lysophosphatidic acid receptor (ATX-LPAR) signaling axis is pivotal in various clinical conditions, including cancer and autoimmune disorders. This axis promotes tumorigenicity by interacting with the tumor microenvironment, facilitating metastasis, and conceding antitumor immunity, thereby fostering resistance to conventional cancer therapies. Recent studies highlight the promise of ATX/LPAR inhibitors in combination with conventional chemotherapeutic drugs to overcome some forms of this resistance, representing a novel therapeutic strategy. In the current study, we employed structure-based virtual screening, integrating pharmacophore modeling and molecular docking, to identify MolPort-137 as a novel ATX inhibitor with an IC50 value of 1.6 ± 0.2 μM in an autotaxin enzyme inhibition assay. Molecular dynamics simulations and binding free energy calculations elucidated the binding mode of MolPort-137 and its critical amino acid interactions. Remarkably, MolPort-137 exhibited no cytotoxicity as a single agent but enhanced the effectiveness of paclitaxel in 4T1 murine breast carcinoma cells and resensitized taxol-resistant cells to paclitaxel treatment, which highlights its potential in combination therapy.

Deoxyhypusine synthase (DHPS) is an enzyme encoded by the DHPS gene, with high expression in various cancers, including ovarian cancer (OC). DHPS regulates the translation initiation factor EIF5A, and EIF5A2 knockout inhibits OC tumor growth and metastasis by blocking the epithelial-to-mesenchymal transition (EMT) and the TGFβ pathway. In this study, we show that DHPS is amplified in OC patients, and its elevated expression correlates with poor survival. Using lentiviral CRISPR/Cas9 vectors for DHPS knockout, we observed EMT inhibition in SKOV3 and OVCAR8 cells through suppressed hypusination and reduced EIF5A2 expression. Inhibition of DHPS activity with GC7 similarly blocked hypusination and EMT. Disrupting DHPS expression, either genetically or pharmacologically, inhibited primary tumor growth and metastasis in OC mouse models. These findings suggest that targeting DHPS and inhibiting hypusination could be promising strategies for OC treatment.

Autotaxin (ATX) is a lysophospholipase D that generates lysophosphatidic acid (LPA) and regulates cancer metastasis, therapeutic resistance, and tumor immunity. We found that myeloid cells in human melanoma biopsies abundantly express ATX and investigated its role in modulating innate tumor immunity using two models of melanoma metastasis-spontaneous and experimental. Targeted knockout of ATX in LysM+ myeloid cells in mice (LysM-KO) reduced both spontaneous and experimental B16-F10 melanoma metastases by >= 50%. Immunoprofiling revealed differences in M2-like alveolar macrophages, neutrophils and regulatory T cells in the metastatic lungs of LysM-WT versus LysM-KO that are model-dependent. These differences extend systemically, with LysM-KO mice bearing experimental metastasis having fewer neutrophils in the spleen than LysM-WT mice. Our results show that (1) LysM+ myeloid cells are an important source of ATX/LPA that promote melanoma metastasis by altering innate tumor immunity, and (2) intratumor and systemic immune profiles vary dynamically during disease progression and are model- dependent.

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-1β, 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.

We have previously reported that, in aortic rings, 18:1 lysophosphatidic acid (LPA) can induce both vasodilation and vasoconstriction depending on the integrity of the endothelium. The predominant molecular species generated in blood serum are poly-unsaturated LPA species, yet the vascular effects of these species are largely unexplored. We aimed to compare the vasoactive effects of seven naturally occurring LPA species in order to elucidate their potential pathophysiological role in vasculopathies. Vascular tone was measured using myography, and thromboxane A2 (TXA2) release was detected by ELISA in C57Bl/6 mouse aortas. The Ca2+-responses to LPA-stimulated primary isolated endothelial cells were measured by Fluo-4 AM imaging. Our results indicate that saturated molecular species of LPA elicit no significant effect on the vascular tone of the aorta. In contrast, all 18 unsaturated carbon-containing (C18) LPAs (18:1, 18:2, 18:3) were effective, with 18:1 LPA being the most potent. However, following inhibition of cyclooxygenase (COX), these LPAs induced similar vasorelaxation, primarily indicating that the vasoconstrictor potency differed among these species. Indeed, C18 LPA evoked a similar Ca2+-signal in endothelial cells, whereas in endothelium-denuded aortas, the constrictor activity increased with the level of unsaturation, correlating with TXA2 release in intact aortas. COX inhibition abolished TXA2 release, and the C18 LPA induced vasoconstriction. In conclusion, polyunsaturated LPA have markedly increased TXA2-releasing and vasoconstrictor capacity, implying potential pathophysiological consequences in vasculopathies.

The development of drug resistance in cancer cells poses a significant challenge for treatment, with nearly 90% of cancer-related deaths attributed to it. Over 50% of ovarian cancer patients and 30–40% of breast cancer patients exhibit resistance to therapies such as Taxol. Previous literature has shown that cytotoxic cancer therapies and ionizing radiation damage tumors, prompting cancer cells to exploit the autotaxin (ATX)–lysophosphatidic acid (LPA)–lysophosphatidic acid receptor (LPAR) signaling axis to enhance survival pathways, thus reducing treatment efficacy. Therefore, targeting this signaling axis has become a crucial strategy to overcome some forms of cancer resistance. Addressing this challenge, we identified and assessed ATX-1d, a novel compound targeting ATX, through computational methods and in vitro assays. ATX-1d exhibited an IC50 of 1.8 ± 0.3 μM for ATX inhibition and demonstrated a significant binding affinity for ATX, as confirmed by MM-GBSA, QM/MM-GBSA, and SAPT in silico methods. ATX-1d significantly amplified the potency of paclitaxel, increasing its effectiveness tenfold in 4T1 murine breast carcinoma cells and fourfold in A375 human melanoma cells without inducing cytotoxic effects as a single agent.

While immune checkpoint inhibitors (ICIs) are promising in the treatment of metastatic melanoma, about half of patients do not respond well to them. Low levels of human leukocyte antigen-DR (HLA-DR) in tumors have been shown to negatively influence prognosis and response to ICIs. Lysophosphatidic acid (LPA) is produced in large amounts by melanoma and is abundantly present in the tumor microenvironment. LPA induces the release of various cytokines and chemokines from tumor cells, which affect cancer development, metastasis, and tumor immunity. In the present study, we investigated the role of LPA-induced IL-10 release in regulating HLA-DR expression and the underlying mechanisms in human melanoma cells. We showed that LPA (0.001–10 μM) dose-dependently increased DR6 transcript levels through activating LPAR1 in HEK293T cells. Knockdown of NF-κB1 abrogated the LPA-increased DR6 expression without affecting basal DR6 expression in both A2058 and A375 melanoma cell lines. LPA (10 µM) significantly increased IL-10 transcripts in A2058 and A375 melanoma cells, the effect was abolished by pharmacological inhibition of LPAR1 or knockdown of DR6. We found a statistically significant correlation between the expression of LPAR1, DR6 and IL-10 in human melanoma tissue and an association between increased expression of LPAR1 and reduced effectiveness of ICI therapy. We demonstrated that LPA (10 µM) markedly suppressed HLA-DR expression in both A375 and A2058 melanoma cells via activating the LPAR1-DR6-IL-10 pathway. These data suggest that the LPAR1-DR6-IL-10 autocrine loop could constitute a novel mechanism used by tumor cells to evade immunosurveillance by decreasing HLA-DR expression.

Supplementary Figure 3. (A) ATX and LPA receptor profiling in B16F10 cells transduced with GFP lentivirus using quantitative real-time PCR. Data are expressed as mean {plus minus} SD of an experiment performed twice in quadruplicates. (B) Representative images of lungs harvested at day 21 from mice that were injected with PBS (left panel, control) and mice that were injected with 1 x 105 GFP-expressing B16F10 cells (right panel). Arrow denotes a single fluorescent tumor nodule on lung surface. Scale bar = 200 microns.

Supplementary Table 1. Primer sequences used in quantitative Real-Time PCR analysis.

Sphingolipids are important biological mediators both in health and disease. We investigated the vascular effects of enhanced sphingomyelinase (SMase) activity in a mouse model of type 2 diabetes mellitus (T2DM) to gain an understanding of the signaling pathways involved. Myography was used to measure changes in the tone of the thoracic aorta after administration of 0.2 U/mL neutral SMase in the presence or absence of the thromboxane prostanoid (TP) receptor antagonist SQ 29,548 and the nitric oxide synthase (NOS) inhibitor L-NAME. In precontracted aortic segments of non-diabetic mice, SMase induced transient contraction and subsequent weak relaxation, whereas vessels of diabetic (Leprdb/Leprdb, referred to as db/db) mice showed marked relaxation. In the presence of the TP receptor antagonist, SMase induced enhanced relaxation in both groups, which was 3-fold stronger in the vessels of db/db mice as compared to controls and could not be abolished by ceramidase or sphingosine-kinase inhibitors. Co-administration of the NOS inhibitor L-NAME abolished vasorelaxation in both groups. Our results indicate dual vasoactive effects of SMase: TP-mediated vasoconstriction and NO-mediated vasorelaxation. Surprisingly, in spite of the general endothelial dysfunction in T2DM, the endothelial NOS-mediated vasorelaxant effect of SMase was markedly enhanced.

Supplemental Figure Legends. Supplemental Figure Legends

Supplementary Figure 5. ATX and LPA receptor profiling in isolated rat alveolar type I-like (A) or type II (B) epithelial cells. Note that expression levels in type II alveolar cells are approximately 10-fold lower than in type I-like cells. Data presented are representative of an experiment performed three times in quadruplicates and are expressed as mean {plus minus} SD.

Introduction:The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5).Methods:Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels.Results:Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation.Conclusion:These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

Supplementary Methods, Figures 1-3, Table 1 from Dual Activity Lysophosphatidic Acid Receptor Pan-Antagonist/Autotaxin Inhibitor Reduces Breast Cancer Cell Migration In vitro and Causes Tumor Regression In vivo

Supplementary Figure 4. Effect of BMP22 on the basal invasion of B16F10 cells across a matrigel layer. Data are representative of an experiment performed twice in quadruplicates and are expressed as mean {plus minus} SD. *Denotes p value < 0.05 using one-way ANOVA followed by a Bonferroni post-test.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator that regulates a variety of cellular functions such as cell proliferation, migration, survival, calcium mobilization, cytoskeletal rearrangements, and neurite retraction. The biological actions of LPA are mediated by at least six G protein-coupled receptors known as LPAR1–6. Given that LPAR1–3 were among the first LPARs identified, the majority of research efforts have focused on understanding their biology. This review provides an in-depth discussion of LPAR5, which has recently emerged as a key player in regulating normal intestinal homeostasis and modulating pathological conditions such as pain, itch, inflammatory diseases, and cancer. We also present a chronological overview of the efforts made to develop compounds that target LPAR5 for use as tool compounds to probe or validate LPAR5 biology and therapeutic agents for the treatment of inflammatory diseases and cancer.

Supplementary Figure 1. Time course of ATX activity measured in the conditioned media (CM) of (A) MM1 rat hepatocarcinoma, (B) HUVEC, (C) RLMVEC, and (D) isolated murine mesothelial cells. Control represents media that were not exposed to cells. Data are expressed as mean {plus minus} SD of an experiment performed three times in triplicates.

Dysregulation of the autotaxin (ATX, Enpp2)‐lysophosphatidic acid (LPA) signaling in cancerous cells contributes to tumorigenesis and therapy resistance. We previously found that ATX activity was elevated in p53‐KO mice compared to wild‐type (WT) mice. Here, we report that ATX expression was upregulated in mouse embryonic fibroblasts from p53‐KO and p53R172H mutant mice. ATX promoter analysis combined with yeast one‐hybrid testing revealed that WT p53 directly inhibits ATX expression via E2F7. Knockdown of E2F7 reduced ATX expression and chromosome immunoprecipitation showed that E2F7 promotes Enpp2 transcription through cooperative binding to two E2F7 sites (promoter region −1393 bp and second intron 996 bp). Using chromosome conformation capture, we found that chromosome looping brings together the two E2F7 binding sites. We discovered a p53 binding site in the first intron of murine Enpp2, but not in human ENPP2. Binding of p53 disrupted the E2F7‐mediated chromosomal looping and repressed Enpp2 transcription in murine cells. In contrast, we found no disruption of E2F7‐mediated ENPP2 transcription via direct p53 binding in human carcinoma cells. In summary, E2F7 is a common transcription factor that upregulates ATX in human and mouse cells but is subject to steric interference by direct intronic p53 binding only in mice.