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Fahed A Elian,1 Ubah Are,1 Sunita Ghosh,2,3 Paulo Nuin,1 Tim Footz,1 Todd PW McMullen,4 David N Brindley,5,6 Michael A Walter1 1Department of Medical Genetics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada; 2Department of Medical Oncology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada; 3Department of Mathematical and Statistical Sciences, Faculty of Science, University of Alberta, Edmonton, AB, Canada; 4Department of Surgery, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada; 5Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada; 6Cancer Research Institute of Northern Alberta, University of Alberta, Edmonton, AB, CanadaCorrespondence: Michael A WalterDepartment of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, 8-32 Medical Sciences Building, Edmonton, AB, T6G 2H7, CanadaTel +1 (780) 492-3028Email mwalter@ualberta.caPurpose: Forkhead box Q1 (FOXQ1) has been shown to contribute to the development and progression of cancers, including ovarian and breast cancer (BC). However, research exploring FOXQ1 expression, copy number variation (CNV), and prognostic value across different BC subtypes is limited. Our purpose was to evaluate FOXQ1 mRNA expression, CNV, and prognostic value across BC subtypes.Materials and Methods: We determined FOXQ1 expression and CNV in BC patient tumors using RT-qPCR and qPCR, respectively. We also analyzed FOXQ1 expression and CNV in BC cell lines in the CCLE database using K-means clustering. The prognostic value of FOXQ1 expression in the TCGA-BRCA database was assessed using univariate and multivariate Cox’s regression analysis as well as using the online tools OncoLnc, GEPIA, and UALCAN.Results: Our analyses reveal that FOXQ1 mRNA is differentially expressed between different subtypes of BC and is significantly decreased in luminal BC and HER2 patients when compared to normal breast tissue samples. Furthermore, analysis of BC cell lines showed that FOXQ1 mRNA expression was independent of CNV. Moreover, patients with low FOXQ1 mRNA expression had significantly poorer overall survival compared to those with high FOXQ1 mRNA expression. Finally, low FOXQ1 expression had a critical impact on the prognostic values of BC patients and was an independent predictor of overall survival when it was adjusted for BC subtypes and to two other FOX genes, FOXF2 and FOXM1.Conclusion: Our study reveals for the first time that FOXQ1 is differentially expressed across BC subtypes and that low expression of FOXQ1 is indicative of poor prognosis in patients with BC.Keywords: transcription factors, mRNA expression, copy number variation, predictive marker

Lysophosphatidate (LPA)-mediated signaling is a vital component of physiological wound healing, but the pathway is subverted to mediate chronic inflammatory signaling in many pathologies, including cancers. LPA, as an extracellular signaling molecule, is produced by the enzyme autotaxin (ATX, gene name ENPP2 ) and signals through six LPA receptors (LPARs). Its signaling is terminated by turnover via the ecto-activity of three lipid phosphate phosphatases (LPPs, gene names PLPP1-3 ). Many pharmacological developments against the LPA-signaling axis are underway, primarily against ATX. An ATX inhibitor against pancreatic ductal adenocarcinoma (PDAC), a very aggressive disease with limited systemic therapeutic options, is currently in clinical trials, and represents the first in-class drug against LPA signaling in cancers. In the present study, we surveyed the expression of ATX, LPARs, and LPPs in human PDACs and their clinical outcomes in two large independent cohorts, the Cancer Genome Atlas (TCGA) and GSE21501. Correlation among gene expressions, biological function and the cell composition of the tumor microenvironment were analysed using gene set enrichment analysis and cell cyber-sorting with xCell. ENPP2, LPAR1, LPAR4, LPAR5, LPAR6, PLPP1 , and PLPP2 were significantly elevated in PDACs compared to normal pancreatic tissue, whereas LPAR2, LPAR3 , and PLPP3 where downregulated (all P≤0.003). Only ENPP2 demonstrated survival differences, with overall survival favoring ENPP2 -high patients (hazard ration 0.5-0.9). ENPP2 was also the only gene with enriched gene patterns for inflammatory and tissue repair gene sets. Epithelial (cancer) cells had increased LPAR2, LPAR5 and PLPP2 expression, and decreased ENPP2, LPAR1, PLPP1 , and PLPP3 gene expression (all P<0.02). Tumor fibroblasts had increased ENPP2, LPAR2, LPAR4, PLPP1 , and PLPP3 expression and decreased LPAR2, LPAR5 , and PLPP2 expression in both cohorts (all P≤0.01). Immune cell populations were not well correlated to gene expression in PDACs, but across both cohorts, cytolytic scores were increased in high-expressing ENPP2, LPAR1, LPAR6, PLPP1 , and PLPP3 tumors (P<0.01). Overall, in PDACs, ENPP2 may switch from an anti-to-pro tumor promoting gene with disease progression. LPAR2 and PLPP2 inhibition are also predicted to have potential therapeutic utility. Future multi-omics investigations are necessarily to validate which LPA signaling components are high-value candidates for pharmacological manipulation in PDAC treatment., Competing Interests: D.N.B. is a member of the Scientific and Clinical Advisory Boards of iOnctura and receives funding for a postdoctoral fellow from iOnctura to work on IOA-289., (AJCR Copyright © 2024.)

Metastasis remains a major challenge in treating breast cancer. Breast tumors metastasize to organ-specific locations such as the brain, lungs, and bone, but why some organs are favored over others remains unclear. Breast tumors also show heterogeneity, plasticity, and distinct microenvironments. This contributes to treatment failure and relapse. The interaction of breast cancer cells with their metastatic microenvironment has led to the concept that primary breast cancer cells act as seeds, whereas the metastatic tissue microenvironment (TME) is the soil. Improving our understanding of this interaction could lead to better treatment strategies for metastatic breast cancer. Targeted treatments for different subtypes of breast cancers have improved overall patient survival, even with metastasis. However, these targeted treatments are based upon the biology of the primary tumor and often these patients' relapse, after therapy, with metastatic tumors. The advent of immunotherapy allowed the immune system to target metastatic tumors. Unfortunately, immunotherapy has not been as effective in metastatic breast cancer relative to other cancers with metastases, such as melanoma. This review will describe the heterogeneic nature of breast cancer cells and their microenvironments. The distinct properties of metastatic breast cancer cells and their microenvironments that allow interactions, especially in bone and brain metastasis, will also be described. Finally, we will review immunotherapy approaches to treat metastatic breast tumors and discuss future therapeutic approaches to improve treatments for metastatic breast cancer.

Overcoming and preventing cancer therapy resistance is the most pressing challenge in modern breast cancer management. Consequently, most modern breast cancer research is aimed at understanding and blocking these therapy resistance mechanisms. One increasingly promising therapeutic target is the autotaxin (ATX)-lysophosphatidate (LPA)-lipid phosphate phosphatase (LPP) axis. Extracellular LPA, produced from albumin-bound lysophosphatidylcholine by ATX and degraded by the ecto-activity of the LPPs, is a potent cell-signaling mediator of tumor growth, invasion, angiogenesis, immune evasion, and resistance to cancer treatment modalities. LPA signaling in the post-natal organism has central roles in physiological wound healing, but these mechanisms are subverted to fuel pathogenesis in diseases that arise from chronic inflammatory processes, including cancer. Over the last 10 years, our understanding of the role of LPA signaling in the breast tumor microenvironment has begun to mature. Tumor-promoting inflammation in breast cancer leads to increased ATX production within the tumor microenvironment. This results in increased local concentrations of LPA that are maintained in part by decreased overall cancer cell LPP expression that would otherwise more rapidly break it down. LPA signaling through six G-protein-coupled LPA receptors expressed by cancer cells can then activate virtually every known tumorigenic pathway. Consequently, to target therapy resistance and tumor growth mediated by LPA signaling, multiple inhibitors against the LPA signaling axis are entering clinical trials. In this review, we summarize recent developments in LPA breast cancer biology, and illustrate how these novel therapeutics against the LPA signaling pathway may be excellent adjuncts to extend the efficacy of evolving breast cancer treatments., Competing Interests: The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article. D.N.B. is a member of the Scientific and Clinical Advisory Boards of iOnctura and receives funding for a postdoctoral fellow from iOnctura to work on an ATX inhibitor, but he has no commercial interests in the company. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results., (Copyright 2024, Benesch et al.)

Tumor microenvironment (TME) of solid tumors including breast cancer is complex and contains a distinct cytokine pattern including TNF-α, which determines the progression and metastasis of breast tumors. The metastatic potential of triple negative breast cancer subtypes is high as compared to other subtypes of breast cancer. NF-κB is key transcription factor regulating inflammation and mitochondrial bioenergetics including oxidative phosphorylation (OXPHOS) genes which determine its oxidative capacity and generating reducing equivalents for synthesis of key metabolites for proliferating breast cancer cells. The differential metabolic adaptation and OXPHOS function of breast cancer subtypes in inflammatory conditions and its contribution to metastasis is not well understood. Here we demonstrated that different subunits of NF-κB are differentially expressed in subtypes of breast cancer patients. RELA, one of the major subunits in regulation of the NF-κB pathway is positively correlated with high level of TNF-α in breast cancer patients. TNF-α induced NF-κB regulates the expression of LYRM7, an assembly factor for mitochondrial complex III. Downregulation of LYRM7 in MDA-MB-231 cells decreases mitochondrial super complex assembly and enhances ROS levels, which increases the invasion and migration potential of these cells. Further, in vivo studies using Infliximab, a monoclonal antibody against TNF-α showed decreased expression of LYRM7 in tumor tissue. Large scale breast cancer databases and human patient samples revealed that LYRM7 levels decreased in triple negative breast cancer patients compared to other subtypes and is determinant of survival outcome in patients. Our results indicate that TNF-α induced NF-κB is a critical regulator of LYRM7, a major factor for modulating mitochondrial functions under inflammatory conditions, which determines growth and survival of breast cancer cells., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

An inflammatory milieu in the tumor microenvironment leads to immune evasion, resistance to cell death, metastasis and poor prognosis in breast cancer patients. TNF-α is a proinflammatory cytokine that regulates multiple aspects of tumor biology from initiation to progression. TNF-α-induced NF-κB activation initiates inflammatory pathways, which determine cell survival, death and tumor progression. One candidate pathway involves the increased secretion of autotaxin, which produces lysophosphatidate that signals through six G-protein-coupled receptors. Significantly, autotaxin is one of the 40-50 most upregulated genes in metastatic tumors. In this study, we investigated the effects of TNF-α by blocking its action with a monoclonal antibody, Infliximab, and studied the effects on autotaxin secretion and tumor progression. Infliximab had little effect on tumor growth, but it decreased lung metastasis by 60% in a syngeneic BALB/c mouse model using 4T1 breast cancer cells. Infliximab-treated mice also showed a decrease in proliferation and metastatic markers like Ki-67 and vimentin in tumors. This was accompanied by decreases in NF-κB activation, autotaxin expression and the concentrations of plasma and tumor cytokines/chemokines which are involved in metastasis. We also demonstrated a positive correlation of TNF-α -NF-κB and ATX expression in breast cancer patients using cancer databases. Studies in vitro showed that TNF-α-induced NF-κB activation increases autotaxin expression and the clone forming ability of 4T1 breast cancer cells. This report highlights the potential role of Infliximab as an additional approach to attenuate signaling through the autotaxin-lysophosphatidate-inflammatory cycle and decrease mortality from metastatic cancer.

Autotaxin (ATX) is a secreted enzyme that produces extracellular lysophosphatidate in physiological wound healing. ATX is overexpressed in many cancers to promote growth, metastasis, and treatment resistance. However, ATX expression is very low in breast cancer cells, and is instead secreted by the tumor microenvironment (TME). Paracrine ATX expression, and its effects on tumor progression, has not been robustly studied in human breast tumors. In this study, ATX expression was analyzed in over 5000 non-metastatic breast cancers from databases TCGA, METABRIC and GSE96058, dichotomized by the median. Gene set enrichment analysis (GSEA) and the xCell algorithm investigated biological functions of ATX and correlation to TME cell populations. TME ATX production was verified by single cell RNA sequencing. The highest ATX expression occurred in endothelial cells and cancer-associated fibroblasts ( P <0.0001). High tumor ATX expression correlated to increased adipocyte, fibroblast, and endothelial cell fractions ( P <0.01), and GSEA demonstrated enriched immune system, tumor suppressor, pro-survival, stemness, and pro-inflammatory signaling in multiple gene sets. Tumor mutational burden was decreased, Ki67 scores were decreased, tumor infiltrating immune cell populations increased, and immune cytolytic activity scores increased (all P <0.01) for ATX-high tumors. Overall survival trends favored ATX-high tumors (hazard ratios 0.75-0.80). In summary, in human breast cancers, ATX is produced by the TME, and in non-metastatic tumors, high levels correlate with an anti-tumor phenotype. Because pre-clinical models use aggressive pro-metastatic cell lines where ATX-mediated signaling promotes tumorigenesis, further research is required to verify an anti-to-pro-tumor phenotype switch with breast cancer progression and/or treatment resistance., Competing Interests: None., (AJCR Copyright © 2023.)

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.

Breast cancer cells produce negligible quantities of autotaxin. Instead, previous work indicated that adipocytes in the inflamed adipose tissue adjacent to breast tumors are a major source of autotaxin secretion that drives breast tumor growth, metastasis, and the loss of efficacy for chemotherapy and radiotherapy. To test this hypothesis, we used mice with an adipocyte-specific knock out of autotaxin. The lack of autotaxin secretion from adipocytes failed to decrease the growth of orthotopic E0771 breast tumors in syngeneic C57BL/6 mice and the growth and lung metastasis of spontaneous breast tumors in MMTV-PyMT mice. However, the inhibition of autotaxin with IOA-289 decreased the growth of E0771 tumors, indicating that another source of autotaxin is responsible for tumor growth. Tumor-associated fibroblasts and leukocytes produce the majority of autotoxin transcripts in the E0771 breast tumors, and we hypothesize that they are the main sources of ATX that drive breast tumor growth. Autotaxin inhibition with IOA-289 increased the numbers of CD8α + -T-cells in the tumors. This was accompanied by decreases in the concentrations of CXCL10, CCL2, and CXCL9 in the plasma and LIF, TGFβ1, TGFβ2, and prolactin in the tumors. Bioinformatics analysis of human breast tumor databases showed that autotaxin (ENPP2) is expressed mainly in endothelial cells and fibroblasts. Autotaxin expression correlated significantly with increases in IL-6 cytokine receptor ligand interactions, signaling by LIF, TGFβ, and prolactin. This confirms the relevance of results from autotaxin inhibition in the mouse model. We propose that inhibiting autotaxin activity that is derived from cells presenting breast tumors such as fibroblasts, leukocytes, or endothelial cells changes the tumor micro-environment in such a way as to inhibit tumor growth., Competing Interests: D.N.B. is a member of the Scientific and Clinical Advisory Boards for iOnctura and receives research funding for a postdoctoral fellow through a MITACS award. M.A.D. is employee and shareholder of iOnctura.

The LPP family is comprised of three enzymes that dephosphorylate bioactive lipid phosphates both intracellularly and extracellularly. Pre-clinical breast cancer models have demonstrated that decreased LPP1/3 with increased LPP2 expression correlates to tumorigenesis. This though has not been well verified in human specimens. In this study, we correlate LPP expression data to clinical outcomes in over 5000 breast cancers from three independent cohorts (TCGA, METABRIC, and GSE96058), investigate biological function using gene set enrichment analysis (GSEA) and the xCell cell-type enrichment analysis, and confirm sources of LPP production in the tumor microenvironment (TME) using single-cell RNA-sequencing (scRNAseq) data. Decreased LPP1/3 and increased LPP2 expression correlated to increased tumor grade, proliferation, and tumor mutational burden (all p < 0.001), as well as worse overall survival (hazard ratios 1.3-1.5). Further, cytolytic activity was decreased, consistent with immune system invasion. GSEA data demonstrated multiple increased inflammatory signaling, survival, stemness, and cell signaling pathways with this phenotype across all three cohorts. scRNAseq and the xCell algorithm demonstrated that most tumor LPP1/3 was expressed by endothelial cells and tumor-associated fibroblasts and LPP2 by cancer cells (all p < 0.01). Restoring the balance in LPP expression levels, particularly through LPP2 inhibition, could represent novel adjuvant therapeutic options in breast cancer treatment.

LPP2 is one of three enzymes in the lipid phosphate phosphatase family (LPP1-3) that dephosphorylate extracellular and intracellular bioactive lipid phosphates and pyrophosphates. LPP2 increases cell growth and LPP2 expression is elevated in a variety of malignancies, implying that LPP2 is a pro-tumorigenic factor. Methods: LPP2 expression in human breast tumors and normal breast tissue was measured by qPCR. To understand the role of LPP2, we knocked out its expression in multiple cell lines using CRISPR/Cas9. Cell proliferation and migration were compared between wild type and LPP2 knockout cells. Cell cycle was measured by flow cytometry, and cell cycle proteins were determined by western blotting. Effects of LPP2 on tumor growth were investigated using syngeneic and xenograft mouse breast cancer models. Results: LPP2 mRNA levels were higher in ER/PR positive, ER/HER2 positive, and triple negative human breast tumors, relative to normal breast tissue. Higher levels of LPP2 in breast tumors, hepatocellular carcinoma, pancreatic adenocarcinoma, and melanomas were prognostic of poorer survival. LPP2 mRNA expression is also increased in Hs-578T, MDA-MB-231, MCF7 and MDA-MB-468 breast cancer cell lines, relative to non-malignant Hs-578Bst, MCF10A and MCF-12A cells. LPP2 knockout in breast cancer cells decreased cell growth by inhibiting G1/S transition, whereas, increasing LPP2 levels in Hs-578Bst and MCF10A cells promoted proliferation. The effects of LPP2 on cell cycle were associated with changes in cyclin A2, cyclin B1, and cell cycle inhibitors, p27 or p21. The level of c-Myc was downregulated by knocking out LPP2, and it was partly restored by re-expressing LPP2. The positive correlation between the expression of LPP2 and c-Myc exists in multiple cancer cell lines including breast, lung, upper aerodigestive tract and urinary tract cancer. LPP2 knockout in MDA-MB-231 or 4T1 cells suppressed tumor formation in mouse breast cancer models, and decreased the in vivo expression of Ki67 and c-Myc of the cancer cells. Conclusion: Targeting LPP2 could provide a new strategy for decreasing c-Myc expression and tumor growth., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Human cytomegalovirus (HCMV) infects 40-70% of adults in developed countries. Detection of HCMV DNA and/or proteins in breast tumors varies considerably, ranging from 0-100%. In this study, nested PCR to detect HCMV glycoprotein B (gB) DNA in breast tumors was shown to be sensitive and specific in contrast to the detection of DNA for immediate early genes. HCMV gB DNA was detected in 18.4% of 136 breast tumors while 62.8% of 94 breast cancer patients were seropositive for HCMV. mRNA for the HCMV immediate early gene was not detected in any sample, suggesting viral latency in breast tumors. HCMV seropositivity was positively correlated with age, body mass index and menopause. Patients who were HCMV seropositive or had HCMV DNA in their tumors were 5.61 (CI 1.77-15.67, p = 0.003) or 5.27 (CI 1.09-28.75, p = 0.039) times more likely to develop Stage IV metastatic tumors, respectively. Patients with HCMV DNA in tumors experienced reduced relapse-free survival ( p = 0.042). Being both seropositive with HCMV DNA-positive tumors was associated with vascular involvement and metastasis. We conclude that determining the seropositivity for HCMV and detection of HCMV gB DNA in the breast tumors could identify breast cancer patients more likely to develop metastatic cancer and warrant special treatment.

Three-dimensional (3D) cell culture technology is a powerful tool in cancer research and drug development. It retains several critical components of the in vivo environment, including cell-cell and cell-extracellular matrix (ECM) interactions in a 3D fashion, gradients of oxygen, nutrients and metabolic waste, and it is thus more physiologically relevant than traditional two-dimensional (2D) cell culture. Here, we describe a simple and versatile method using commercially available chamber slides and Matrigel, a surrogate ECM hydrogel, to set up a 3D culture model for breast cancer cells. In this 3D culture model, cells form aggregates or spheroids on top of a thin layer of Matrigel, which can be fixed directly onto the chamber slides for cell imaging and immunofluorescence staining. Alternatively, RNA and protein can be extracted from the cells for further investigation. This 3D cell culture model provides a useful platform for cancer research and drug development, in which the effects of novel compounds or genetic modifications can be tested., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Human cytomegalovirus (HCMV) infects 40-70% of adults in developed countries. HCMV proteins and DNA are detected in tumors and metastases, suggesting an association with increased invasion. We investigated HCMV infection in human breast cancer cell lines compared to fibroblasts, a component of tumors, and the role of platelet-derived growth factor receptor-α (PDGFRα). HCMV productively infected HEL299 fibroblasts and, to a lesser extent, Hs578T breast cancer cells. Infection of another triple-negative cell line, MDA-MB-231, and also MCF-7 cells, was extremely low. These disparate infection rates correlated with expression of PDGFRA , which facilitates HCMV uptake. Increasing PDGFRA expression in T-47D breast cancer and BCPAP thyroid cancer cells markedly increased HCMV infection. Conversely, HCMV infection decreased PDGFRA expression, potentially attenuating signaling through this receptor. HCMV infection of fibroblasts promoted the secretion of proinflammatory factors, whereas an overall decreased secretion of inflammatory factors was observed in infected Hs578T cells. We conclude that HCMV infection in tumors will preferentially target tumor-associated fibroblasts and breast cancer cells expressing PDGFRα. HCMV infection in the tumor microenvironment, rather than cancer cells, will increase the inflammatory milieu that could enhance metastasis involving lysophosphatidate.

Autotaxin (ATX) is a secreted enzyme responsible for producing lysophosphatidic acid (LPA). The ATX/LPA signaling axis is typically activated in wound healing and tissue repair processes. The ATX/LPA axis is highjacked and upregulated in the progression and persistence of several chronic inflammatory diseases, including cancer. As ATX inhibitors are now progressing to clinical testing, innovative diagnostic tools such as positron emission tomography (PET) are needed to measure ATX expression in vivo accurately. The radiotracer, [ 18 F]PRIMATX, was recently developed and tested for PET imaging of ATX in vivo in a murine melanoma model. The goal of the present work was to further validate [ 18 F]PRIMATX as a PET imaging agent by analyzing its in vivo metabolic stability and suitability for PET imaging of ATX in models of human 8305C thyroid tumor and murine 4T1 breast cancer. [ 18 F]PRIMATX displayed favorable metabolic stability in vivo (65% of intact radiotracer after 60 min p.i.) and provided sufficient tumor uptake profiles in both tumor models. Radiotracer uptake could be blocked by 8-12% in 8305C thyroid tumors in the presence of ATX inhibitor AE-32-NZ70 as determined by PET and ex vivo biodistribution analyses. [ 18 F]PRIMATX also showed high brain uptake, which was reduced by 50% through the administration of ATX inhibitor AE-32-NZ70. [ 18 F]PRIMATX is a suitable radiotracer for PET imaging of ATX in the brain and peripheral tumor tissues.

Signaling by the bioactive sphingolipid, sphingosine 1-phosphate (S1P), and its precursors are emerging areas in pregnancy research. S1P and ceramide levels increase towards end of gestation, suggesting a physiological role in parturition. However, high levels of circulating S1P and ceramide are correlated with pregnancy disorders such as preeclampsia, gestational diabetes mellitus and intrauterine growth restriction. Expression of placental and decidual enzymes that metabolize S1P and S1P receptors are also dysregulated during pregnancy complications. In this review, we provide an in-depth examination of the signaling mechanism of S1P and ceramide in various reproductive tissues during gestation. These factors determine implantation and early pregnancy success by modulating corpus luteum function from progesterone production to luteolysis through to apoptosis. We also highlight the role of S1P through receptor signaling in inducing decidualization and angiogenesis in the decidua, as well as regulating extravillous trophoblast migration to anchor the placenta into the uterine wall. Recent advances on the role of the S1P:ceramide rheostat in controlling the fate of villous trophoblasts and the role of S1P as a negative regulator of trophoblast syncytialization to a multinucleated placental barrier are discussed. This review also explores the role of S1P in anti-inflammatory and pro-inflammatory signaling, its role as a vasoconstrictor, and the effects of S1P metabolizing enzymes and receptors in pregnancy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) promote vasculogenesis, angiogenesis, and wound healing by activating a plethora of overlapping signaling pathways that stimulate mitogenesis, cell survival, and migration. As such, maladaptive signaling by LPA and S1P have major effects in increasing tumor progression and producing poor patient outcomes after chemotherapy and radiotherapy. Many signaling actions of S1P and LPA are not redundant; each are vital in normal physiology and their metabolisms differ. In the present work, we studied how LPA signaling impacts S1P metabolism and signaling in MDA-MB-231 and MCF-7 breast cancer cells. LPA increased sphingosine kinase-1 (SphK1) synthesis and rapidly activated cytosolic SphK1 through association with membranes. Blocking phospholipase D activity attenuated the LPA-induced activation of SphK1 and the synthesis of ABCC1 and ABCG2 transporters that secrete S1P from cells. This effect was magnified in doxorubicin-resistant MCF-7 cells. LPA also facilitated S1P signaling by increasing mRNA expression for S1P 1 receptors. Doxorubicin-resistant MCF-7 cells had increased S1P 2 and S1P 3 receptor expression and show increased LPA-induced SphK1 activation, increased expression of ABCC1, ABCG2 and greater S1P secretion. Thus, LPA itself and LPA-induced S1P signaling counteract doxorubicin-induced death of MCF-7 cells. We conclude from the present and previous studies that LPA promotes S1P metabolism and signaling to coordinately increase tumor growth and metastasis and decrease the effectiveness of chemotherapy and radiotherapy for breast cancer treatment., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

This article is intended to recognize and the life time contribution that Dr. Viswanathan Natarajan has made to the advancement of lipid metabolism and lipid signaling. In particular, his major contributions in the last three decades have been made in understanding how lipids such as phosphatidic acid, lysophosphatidic acid, sphingosine 1-phosphate and cardiolipin contribute to healthy lung functions and to a variety of lung pathologies. We celebrate a truly remarkable career and look forward to seeing even more remarkable discoveries., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

LPIN1 mutations are responsible for inherited recurrent rhabdomyolysis, a life-threatening condition with no efficient therapeutic intervention. Here, we conduct a bedside-to-bench-and-back investigation to study the pathophysiology of lipin1 deficiency. We find that lipin1-deficient myoblasts exhibit a reduction in phosphatidylinositol-3-phosphate close to autophagosomes and late endosomes that prevents the recruitment of the GTPase Armus, locks Rab7 in the active state, inhibits vesicle clearance by fusion with lysosomes, and alters their positioning and function. Oxidized mitochondrial DNA accumulates in late endosomes, where it activates Toll-like receptor 9 (TLR9) and triggers inflammatory signaling and caspase-dependent myolysis. Hydroxychloroquine blocks TLR9 activation by mitochondrial DNA in vitro and may attenuate flares of rhabdomyolysis in 6 patients treated. We suggest a critical role for defective clearance of oxidized mitochondrial DNA that activates TLR9-restricted inflammation in lipin1-related rhabdomyolysis. Interventions blocking TLR9 activation or inflammation can improve patient care in vivo ., Competing Interests: Y.H., F.-X.M., C.L., P.v.E., and P.d.L. have filed PCT (WO/2017/085115; EP3377095; PCT/ EP2016/077843) and US patent applications (US20180325890). Y.H., F.-X.M., M.M., P.v.E., and P.d.L. have filed PCT (WO/2019/020732; PCT/EP2018/070256) patent applications. The remaining authors declare no competing interests., (© 2021 The Authors.)

Extracellular lysophosphatidate (LPA) signalling is regulated by the balance of LPA formation by autotaxin (ATX) versus LPA degradation by lipid phosphate phosphatases (LPP) and by the relative expressions of six G-protein-coupled LPA receptors. These receptors increase cell proliferation, migration, survival and angiogenesis. Acute inflammation produced by tissue damage stimulates ATX production and LPA signalling as a component of wound healing. If inflammation does not resolve, LPA signalling becomes maladaptive in conditions including arthritis, neurologic pain, obesity and cancers. Furthermore, LPA signalling through LPA1 receptors promotes fibrosis in skin, liver, kidneys and lungs. LPA also promotes the spread of tumours to other organs (metastasis) and the pro-survival properties of LPA explain why LPA counteracts the effects of chemotherapeutic agents and radiotherapy. ATX is secreted in response to radiation-induced DNA damage during cancer treatments and this together with increased LPA1 receptor expression leads to radiation-induced fibrosis. The anti-inflammatory agent, dexamethasone, decreases levels of inflammatory cytokines/chemokines. This is linked to a coordinated decrease in the production of ATX and LPA1/2 receptors and increased LPA degradation through LPP1. These effects explain why dexamethasone attenuates radiation-induced fibrosis. Increased LPA signalling is also associated with cardiovascular disease including atherosclerosis and deranged LPA signalling is associated with pregnancy complications including preeclampsia and intrahepatic cholestasis of pregnancy. LPA contributes to chronic inflammation because it stimulates the secretion of inflammatory cytokines/chemokines, which increase further ATX production and LPA signalling. Attenuating maladaptive LPA signalling provides a novel means of treating inflammatory diseases that underlie so many important medical conditions., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Lipid phosphate phosphatases (LPPs) are a group of three enzymes (LPP1-3) that belong to a phospholipid phosphatase (PLPP) family. The LPPs dephosphorylate a wide spectrum of bioactive lipid phosphates, among which lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) are two important extracellular signaling molecules. The LPPs are integral membrane proteins, which are localized on plasma membranes and intracellular membranes, including the endoplasmic reticulum and Golgi network. LPPs regulate signaling transduction in cancer cells and demonstrate different effects in cancer progression through the breakdown of extracellular LPA and S1P and other intracellular substrates. This review is intended to summarize an up-to-date understanding about the functions of LPPs in cancers.

Autotaxin (ATX) is a secreted enzyme that produces lysophosphatidate (LPA), which signals through six G-protein coupled receptors, promoting tumor growth, metastasis, and survival from chemotherapy and radiotherapy. Many cancer cells produce ATX, but breast cancer cells express little ATX. In breast tumors, ATX is produced by tumor-associated stroma. Breast tumors are also surrounded by adipose tissue, which is a major bodily source of ATX. In mice, a high-fat diet increases adipocyte ATX production. ATX production in obesity is also increased because of low-level inflammation in the expanded adipose tissue. This increased ATX secretion and consequent LPA signaling is associated with decreased adiponectin production, which results in adverse metabolic profiles and glucose homeostasis. Increased ATX production by inflamed adipose tissue may explain the obesity-breast cancer association. Breast tumors produce inflammatory mediators that stimulate ATX transcription in tumor-adjacent adipose tissue. This drives a feedforward inflammatory cycle since increased LPA signaling increases production of more inflammatory mediators and cyclooxygenase-2. Inhibiting ATX activity, which has implications in breast cancer adjuvant treatments, attenuates this cycle. Targeting ATX activity and LPA signaling may potentially increase chemotherapy and radiotherapy efficacy, and decrease radiation-induced fibrosis morbidity independently of breast cancer type because most ATX is not derived from breast cancer cells.

Autotaxin (ATX) is a secreted enzyme that hydrolyzes lysophosphatidylcholine to produce lysophosphatidate (LPA), which signals through six G-protein coupled receptors (GPCRs). Signaling through LPA is terminated by its degradation by a family of three lipid phosphate phosphatases (LPPs). LPP1 also attenuates signaling downstream of the activation of LPA receptors and some other GPCRs. The ATX-LPA axis mediates a plethora of activities such as cell proliferation, survival, migration, angiogenesis and inflammation, which perform an important role in facilitating wound healing. This wound healing response is hijacked by cancers where there is decreased expression of LPP1 and LPP3 and increased expression of ATX. This maladaptive regulation of LPA signaling also causes chronic inflammation, which has been recognized as one of the hallmarks in cancer. The increased LPA signaling promotes cell survival and migration and attenuates apoptosis, which stimulates tumor growth and metastasis. The wound healing functions of increased LPA signaling also protect cancer cells from effects of chemotherapy and radiotherapy. In this review, we will summarize knowledge of the ATX-LPA axis and its role in the development of resistance to chemotherapy and radiotherapy. We will also offer insights for developing strategies of targeting ATX-LPA axis as a novel part of cancer treatment. This article is part of a Special Issue entitled Lysophospholipids and their receptors: New data and new insights into their function edited by Susan Smyth, Viswanathan Natarajan and Colleen McMullen., Competing Interests: Declaration of competing interest DNB has received consultancy payments and/or research funding from Ono Pharmaceutical Co. Ltd. (Osaka, Japan) and a research grant from Galapagos NV (Mechelen, Belgium). These groups had no role in the design of the review. The other authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We recently showed that radiation-induced DNA damage in breast adipose tissue increases autotaxin secretion, production of lysophosphatidate (LPA) and expression of LPA 1/2 receptors. We also established that dexamethasone decreases autotaxin production and LPA signaling in non-irradiated adipose tissue. In the present study, we showed that dexamethasone attenuated the radiation-induced increases in autotaxin activity and the concentrations of inflammatory mediators in cultured human adipose tissue. We also exposed a breast fat pad in mice to three daily 7.5 Gy fractions of X-rays. Dexamethasone attenuated radiation-induced increases in autotaxin activity in plasma and mammary adipose tissue and LPA 1 receptor levels in adipose tissue after 48 h. DEX treatment during five daily fractions of 7.5 Gy attenuated fibrosis by ~70% in the mammary fat pad and underlying lungs at 7 weeks after radiotherapy. This was accompanied by decreases in CXCL2, active TGF-β1, CTGF and Nrf2 at 7 weeks in adipose tissue of dexamethasone-treated mice. Autotaxin was located at the sites of fibrosis in breast tissue and in the underlying lungs. Consequently, our work supports the premise that increased autotaxin production and lysophosphatidate signaling contribute to radiotherapy-induced breast fibrosis and that dexamethasone attenuated the development of fibrosis in part by blocking this process.

After a decade of intense preclinical investigations, the first in-class autotaxin inhibitor, GLPG1690, has entered Phase III clinical trials for idiopathic pulmonary fibrosis. In the intervening time, a deeper understanding of the role of the autotaxin-lysophosphatidate (LPA)-lipid phosphate phosphatase axis in breast cancer progression and treatment resistance has emerged. Concordantly, appreciation of the tumor microenvironment and chronic inflammation in cancer biology has matured. The role of LPA as a central mediator behind these concepts has been exemplified within the breast cancer field. In this review, we will summarize current challenges in breast cancer therapy and delineate how blocking LPA signaling could provide novel adjuvant therapeutic options for overcoming therapy resistance and adverse side effects, including radiation-induced fibrosis. The advent of autotaxin inhibitors in clinical practice could herald their applications as adjuvant therapies to improve the therapeutic indexes of existing treatments for breast and other cancers.

Autotaxin catalyzes the formation of lysophosphatidic acid, which stimulates tumor growth and metastasis and decreases the effectiveness of cancer therapies. In breast cancer, autotaxin is secreted mainly by breast adipocytes, especially when stimulated by inflammatory cytokines produced by tumors. In this work, we studied the effects of an ATX inhibitor, GLPG1690, which is in phase III clinical trials for idiopathic pulmonary fibrosis, on responses to radiotherapy and chemotherapy in a syngeneic orthotopic mouse model of breast cancer. Tumors were treated with fractionated external beam irradiation, which was optimized to decrease tumor weight by approximately 80%. Mice were also dosed twice daily with GLPG1690 or vehicle beginning at 1 day before the radiation until 4 days after radiation was completed. GLPG1690 combined with irradiation did not decrease tumor growth further compared with radiation alone. However, GLPG1690 decreased the uptake of 3'-deoxy-3'-[ 18 F]-fluorothymidine by tumors and the percentage of Ki67-positive cells. This was also associated with increased cleaved caspase-3 and decreased Bcl-2 levels in these tumors. GLPG1690 decreased irradiation-induced C-C motif chemokine ligand-11 in tumors and levels of IL9, IL12p40, macrophage colony-stimulating factor, and IFNγ in adipose tissue adjacent to the tumor. In other experiments, mice were treated with doxorubicin every 2 days after the tumors developed. GLPG1690 acted synergistically with doxorubicin to decrease tumor growth and the percentage of Ki67-positive cells. GLPG1690 also increased 4-hydroxynonenal-protein adducts in these tumors. These results indicate that inhibiting ATX provides a promising adjuvant to improve the outcomes of radiotherapy and chemotherapy for breast cancer., (©2019 American Association for Cancer Research.)

Breast cancer patients are usually treated with multiple fractions of radiotherapy (RT) to the whole breast after lumpectomy. We hypothesized that repeated fractions of RT would progressively activate the autotaxin-lysophosphatidate-inflammatory cycle. To test this, a normal breast fat pad and a fat pad containing a mouse 4T1 tumor were irradiated with X-rays using a small-animal "image-guided" RT platform. A single RT dose of 7.5 Gy and three daily doses of 7.5 Gy increased ATX activity and decreased plasma adiponectin concentrations. The concentrations of IL-6 and TNFα in plasma and of VEGF, G-CSF, CCL11 and CXCL10 in the irradiated fat pad were increased, but only after three fractions of RT. In 4T1 breast tumor-bearing mice, three fractions of 7.5 Gy augmented tumor-induced increases in plasma ATX activity and decreased adiponectin levels in the tumor-associated mammary fat pad. There were also increased expressions of multiple inflammatory mediators in the tumor-associated mammary fat pad and in tumors, which was accompanied by increased infiltration of CD45+ leukocytes into tumor-associated adipose tissue. This work provides novel evidence that increased ATX production is an early response to RT and that repeated fractions of RT activate the autotaxin-lysophosphatidate-inflammatory cycle. This wound healing response to RT-induced damage could decrease the efficacy of further fractions of RT.

Metastasis is the leading cause of mortality in breast cancer patients and lysophosphatidate (LPA) signaling promotes this process. LPA signaling is attenuated by lipid phosphate phosphatase-1 (LPP1) whose activity is decreased in cancers. Consequently, increasing LPP1 levels suppresses breast tumor growth and metastasis. This study shows that increasing LPP1 in breast cancer cells decreases transcription through cFos and cJun. This decreases production of cyclin D1/D3 and matrix metalloproteinases (MMPs), which provides new insights into the role of LPP1 in controlling tumor growth and metastasis. Methods : Invasiveness was determined by a Matrigel invasion assay. MMP expression was measured by qPCR, multiplex LASER bead technology and gelatin zymography. Levels of cJUN, cFOS, FRA1, cyclin D1, and cyclin D3 were determined by qPCR and western blotting. Collagen was determined by Picro-Sirius Red staining. Results : Increasing LPP1 expression inhibited invasion of MDA-MB-231 breast cancer cells through Matrigel. This was accompanied by decreases in expression of MMP-1, -3, -7, -9, -10, -12 and -13, which are transcriptionally regulated by the AP-1 complex. Increasing LPP1 attenuated the induction of mRNA of MMP-1, -3, cFOS, and cJUN by EGF or TNFα, but increased FRA1. LPP1 expression also decreased the induction of protein levels for cFOS and cJUN in nuclei and cytoplasmic fractions by EGF and TNFα. Protein levels of cyclin D1 and D3 were also decreased by LPP1. Although FRA1 in total cell lysates or cytoplasm was increased by LPP1, nuclear FRA1 was not affected. LPP1-induced decreases in MMPs in mouse tumors created with MDA-MB-231 cells were accompanied by increased collagen in the tumors and fewer lung metastases. Knockdown of LPP1 in MDA-MB-231 cells increased the protein levels of MMP-1 and -3. Human breast tumors also have lower levels of LPP1 and higher levels of cJUN, cFOS, MMP-1, -7, -8, -9, -12, -13, cyclin D1, and cyclin D3 relative to normal breast tissue. Conclusion : This study demonstrated that the low LPP1 expression in breast cancer cells is associated with high levels of cyclin D1/D3 and MMPs as a result of increased transcription by cFOS and cJUN. Increasing LPP1 expression provides a novel approach for decreasing transcription through AP-1, which could provide a strategy for decreasing tumor growth and metastasis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

The sphingolipid and lysophosphatidate regulatory networks impact diverse mechanisms attributed to cancer cells and the tumor immune microenvironment. Deciphering the complexity demands implementation of a holistic approach combined with higher-resolution techniques. We implemented a multi-modular integrative approach consolidating the latest accomplishments in gene expression profiling, prognostic/predictive modeling, next generation digital pathology, and systems biology for epithelial ovarian cancer. We assessed patient-specific transcriptional profiles using the sphingolipid/lysophosphatidate/immune-associated signature. This revealed novel sphingolipid/lysophosphatidate-immune gene-gene associations and distinguished tumor subtypes with immune high/low context. These were characterized by robust differences in sphingolipid-/lysophosphatidate-related checkpoints and the drug response. The analysis also nominates novel survival models for stratification of patients with CD68 , LPAR3 , SMPD1 , PPAP2B , and SMPD2 emerging as the most prognostically important genes. Alignment of proprietary data with curated transcriptomic data from public databases across a variety of malignancies (over 600 categories; over 21,000 arrays) showed specificity for ovarian carcinoma. Our systems approach identified novel sphingolipid-lysophosphatidate-immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes. This holds great promise for delivering novel stratifying and targeting strategies.

Cytomegalovirus (CMV) infects 40⁻70% of women, but infection has been reported in >95% of breast cancer patients. We investigated the consequences of these observations by infecting mice with mCMV or a negative control medium for 4 days, 11 days or 10 weeks to establish active, intermediate or latent infections, respectively. Syngeneic 4T1 or E0771 breast cancer cells were then injected into a mammary fat pad of BALB/c or C57BL/6 mice, respectively. Infection did not affect tumor growth in these conditions, but latently infected BALB/c mice developed more lung metastases. The latent mCMV infection of MMTV-PyVT mice, which develop spontaneous breast tumors, also did not affect the number or sizes of breast tumors. However, there were more tumors that were multilobed with greater blood content, which had enhanced vasculature and decreased collagen content. Most significantly, mCMV infection also increased the number and size of lung metastases, which showed a higher cell proliferation. Viral DNA was detected in breast tumors and lung nodules although viral mRNA was not. These novel results have important clinical implications since an increased metastasis is prognostic of decreased survival. This work provides evidence that treating or preventing HCMV infections may increase the life expectancy of breast cancer patients by decreasing metastasis.

Lysophosphatidate (LPA) signaling through 6 receptors is regulated by the balance of LPA production by autotaxin (ATX) vs. LPA degradation by lipid phosphate phosphatases (LPPs). LPA promotes an inflammatory cycle by increasing the synthesis of cyclooxygenase-2 and multiple inflammatory cytokines that stimulate further ATX production. We aimed to determine whether the anti-inflammatory glucocorticoid (GC) dexamethasone (Dex) functions partly by decreasing the ATX-LPA inflammatory cycle in adipose tissue, a major site of ATX secretion. Treatment of human adipose tissue with 10-1000 nM Dex decreased ATX secretion, increased LPP1 expression, and decreased mRNA expressions of IL-6, TNF-α, peroxisome proliferator-activated receptor (PPAR)-γ, and adiponectin. Cotreatment with rosiglitazone (an insulin sensitizer), insulin, or both abolished Dex-induced decreases in ATX and adiponectin secretion, but did not reverse Dex-induced decreases in secretions of 20 inflammatory cytokines and chemokines. Dex-treated mice exhibited lower ATX activity in plasma, brain, and adipose tissue; decreased mRNA levels for LPA and sphingosine 1-phosphate (S1P) receptors in brain; and decreased plasma concentrations of LPA and S1P. Our results establish a novel mechanism for the anti-inflammatory effects of Dex through decreased signaling by the ATX-LPA-inflammatory axis. The GC action in adipose tissue has implications for the pathogenesis of insulin resistance and obesity in metabolic syndrome and breast cancer treatment.-Meng, G., Tang, X., Yang, Z., Zhao, Y., Curtis, J. M., McMullen, T. P. W., Brindley, D. N. Dexamethasone decreases the autotaxin-lysophosphatidate-inflammatory axis in adipose tissue: implications for the metabolic syndrome and breast cancer.

The lipin phosphatidic acid phosphatase (PAP) enzymes are required for triacylglycerol (TAG) synthesis from glycerol 3-phosphate in most mammalian tissues. The 3 lipin proteins (lipin 1, lipin 2, and lipin 3) each have PAP activity, but have distinct tissue distributions, with lipin 1 being the predominant PAP enzyme in many metabolic tissues. One exception is the small intestine, which is unique in expressing exclusively lipin 2 and lipin 3. TAG synthesis in small intestinal enterocytes utilizes 2-monoacylglycerol and does not require the PAP reaction, making the role of lipin proteins in enterocytes unclear. Enterocyte TAGs are stored transiently as cytosolic lipid droplets or incorporated into lipoproteins (chylomicrons) for secretion. We determined that lipin enzymes are critical for chylomicron biogenesis, through regulation of membrane phospholipid composition and association of apolipoprotein B48 with nascent chylomicron particles. Lipin 2/3 deficiency caused phosphatidic acid accumulation and mammalian target of rapamycin complex 1 (mTORC1) activation, which were associated with enhanced protein levels of a key phospholipid biosynthetic enzyme (CTP:phosphocholine cytidylyltransferase α) and altered membrane phospholipid composition. Impaired chylomicron synthesis in lipin 2/3 deficiency could be rescued by normalizing phospholipid synthesis levels. These data implicate lipin 2/3 as a control point for enterocyte phospholipid homeostasis and chylomicron biogenesis.

A quarter-century after the discovery of autotaxin in cell culture, the autotaxin-lysophosphatidate (LPA)-lipid phosphate phosphatase axis is now a promising clinical target for treating chronic inflammatory conditions, mitigating fibrosis progression, and improving the efficacy of existing cancer chemotherapies and radiotherapy. Nearly half of the literature on this axis has been published during the last five years. In cancer biology, LPA signaling is increasingly being recognized as a central mediator of the progression of chronic inflammation in the establishment of a tumor microenvironment which promotes cancer growth, immune evasion, metastasis, and treatment resistance. In this review, we will summarize recent advances made in understanding LPA signaling with respect to chronic inflammation and cancer. We will also provide perspectives on the applications of inhibitors of LPA signaling in preventing cancer initiation, as adjuncts extending the efficacy of current cancer treatments by blocking inflammation caused by either the cancer or the cancer therapy itself, and by disruption of the tumor microenvironment. Overall, LPA, a simple molecule that mediates a plethora of biological effects, can be targeted at its levels of production by autotaxin, LPA receptors or through LPA degradation by lipid phosphate phosphatases. Drugs for these applications will soon be entering clinical practice., Competing Interests: David N. Brindley has received consultancy payments and/or research funding from Ono Pharmaceuticals Ltd. (Osaka, Japan) and Galapagos NV (Mechelen, Belgium). These groups had no role in the design of the review. The other authors declare no conflicts of interest.

Lipin-1 is a Mg 2+ -dependent phosphatidic acid phosphatase (PAP) that in mice is necessary for normal glycerolipid biosynthesis, controlling adipocyte metabolism, and adipogenic differentiation. Mice carrying inactivating mutations in the Lpin1 gene display the characteristic features of human familial lipodystrophy. Very little is known about the roles of lipin-1 in human adipocyte physiology. Apparently, fat distribution and weight is normal in humans carrying LPIN1 inactivating mutations, but a detailed analysis of adipose tissue appearance and functions in these patients has not been available so far. In this study, we performed a systematic histopathological, biochemical, and gene expression analysis of adipose tissue biopsies from human patients harboring LPIN1 biallelic inactivating mutations and affected by recurrent episodes of severe rhabdomyolysis. We also explored the adipogenic differentiation potential of human mesenchymal cell populations derived from lipin-1 defective patients. White adipose tissue from human LPIN1 mutant patients displayed a dramatic decrease in lipin-1 protein levels and PAP activity, with a concomitant moderate reduction of adipocyte size. Nevertheless, the adipose tissue develops without obvious histological signs of lipodystrophy and with normal qualitative composition of storage lipids. The increased expression of key adipogenic determinants such as SREBP1 , PPARG , and PGC1A shows that specific compensatory phenomena can be activated in vivo in human adipocytes with deficiency of functional lipin-1., (Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.)

Elderly people often suffer adverse health because of inflammation associated with poor metabolism and cardiovascular dysfunction, but these conditions present differently in men and women. We performed experiments in aged male and female mice to understand this sexual dimorphism. We focused on sphingosine 1-phosphate (S1P) signaling, which has both protective and detrimental effects on vascular and metabolic function. We examined vascular function of mesenteric (resistance) arteries from aged male and female wild-type (WT) mice compared to littermate S1P receptor 3 (S1PR3) knockouts (KO). We also measured plasma glucose, insulin, triglycerides, adiponectin, corticosterone and inflammatory cytokines. The novel results of this study are: 1) methacholine-induced vasodilation relied completely on S1PR3 in both sexes, but was dependent on nitric oxide synthase (NOS) only in arteries from aged female mice; 2) S1P-induced vasoconstriction depended solely on S1PR3 in arteries from males, but only partly in females; 3) vasoconstriction to a thromboxane mimetic was decreased by endogenous NOS activity only in arteries from females, regardless of genotype; 4) myogenic responses were lower in arteries from aged WT males compared to females and responses in arteries from KO females were lower than WT females, while the opposite was true of arteries from male mice; 5) aged male mice showed higher fasting glucose and triglycerides with lower plasma adiponectin compared to females and 6) lack of signaling through S1PR3 in females was associated with decreased plasma adiponectin and increased inflammatory mediators. This study showed that there is considerable sexual dimorphism in the vascular and metabolic responses of aged mice and that reduced signaling through S1PR3 could be one mechanism to explain these effects. These results also emphasize that different treatments for mitigating the deleterious effects on vascular health in aged males versus females should be considered., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lysophosphatidate (LPA) is emerging as a potent mediator of cancer progression in the tumor microenvironment. Strategies for targeting LPA signaling have recently entered clinical trials for fibrosis. These therapies have potential to improve the efficacies of existing chemotherapies and radiotherapy by attenuating chronic inflammation, irrespective of diverse mutations within cancer cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We have previously established that adipose tissue adjacent to breast tumors becomes inflamed by tumor-derived cytokines. This stimulates autotaxin (ATX) secretion from adipocytes, whereas breast cancer cells produce insignificant ATX. Lysophosphatidate produced by ATX promotes inflammatory cytokine secretion in a vicious inflammatory cycle, which increases tumor growth and metastasis and decreases response to chemotherapy. We hypothesized that damage to adipose tissue during radiotherapy for breast cancer should promote lysophosphatidic acid (LPA) signaling and further inflammatory signaling, which could potentially protect cancer cells from subsequent fractions of radiation therapy. To test this hypothesis, we exposed rat and human adipose tissue to radiation doses (0.25-5 Gy) that were expected during radiotherapy. This exposure increased mRNA levels for ATX, cyclooxygenase-2, IL-1β, IL-6, IL-10, TNF-α, and LPA 1 and LPA 2 receptors by 1.8- to 5.1-fold after 4 to 48 h. There were also 1.5- to 2.5-fold increases in the secretion of ATX and 14 inflammatory mediators after irradiating at 1 Gy. Inhibition of the radiation-induced activation of NF-κB, cyclooxygenase-2, poly (ADP-ribose) polymerase-1, or ataxia telangiectasia and Rad3-related protein blocked inflammatory responses to γ-radiation. Consequently, collateral damage to adipose tissue during radiotherapy could establish a comprehensive wound-healing response that involves increased signaling by LPA, cyclooxygenase-2, and other inflammatory mediators that could decrease the efficacy of further radiotherapy or chemotherapy.-Meng, G., Tang, X., Yang, Z., Benesch, M. G. K., Marshall, A., Murray, D., Hemmings, D. G., Wuest, F., McMullen, T. P. W., Brindley, D. N. Implications for breast cancer treatment from increased autotaxin production in adipose tissue after radiotherapy., (© FASEB.)

Introduction: One of the main reasons for disease recurrence in the curative breast cancer treatment setting is the development of drug resistance. Microtubule targeted agents (MTAs) are among the most commonly used drugs for the treatment of breaset cancer and therefore overcoming taxane resistance is of primary clinical importance. Our group has previously demonstrated that the microtubule dynamics of docetaxel-resistant MCF-7TXT cells are insensitivity to docetaxel due to the distinct expression profiles of β-tubulin isotypes in addition to the high expression of p-glycoprotein (ABCB1). In the present investigation we examined whether taxane-resistant breast cancer cells are more sensitive to microtubule destabilizing agents including vinca alkaloids and colchicine-site binding agents (CSBAs) than the non-resistant cells., Methods: Two isogenic MCF-7 breast cancer cell lines were selected for resistance to docetaxel (MCF-7TXT) and the wild type parental cell line (MCF-7CC) to examine if taxane-resistant breast cancer cells are sensitive to microtubule-destabilizing agents including vinca alkaloids and CSBAs. Cytotoxicity assays, immunoblotting, indirect immunofluorescence and live imaging were used to study drug resistance, apoptosis, mitotic arrest, microtubule formation, and microtubule dynamics., Results: MCF-7TXT cells were demonstrated to be cross resistant to vinca alkaloids, but were more sensitive to treatment with colchicine compared to parental non-resistant MCF-7CC cells. Cytotoxicity assays indicated that the IC50 of MCF-7TXT cell to vinorelbine and vinblastine was more than 6 and 3 times higher, respectively, than that of MCF-7CC cells. By contrast, the IC50 of MCF-7TXT cell for colchincine was 4 times lower than that of MCF-7CC cells. Indirect immunofluorescence showed that all MTAs induced the disorganization of microtubules and the chromatin morphology and interestingly each with a unique pattern. In terms of microtubule and chromain morphology, MCF-7TXT cells were more resistant to vinorelbine and vinblastine, but more sensitive to colchicine compared to MCF-7CC cells. PARP cleavage assay further demonstrated that all of the MTAs induced apoptosis of the MCF-7 cells. However, again, MCF-7TXT cells were more resistant to vinorelbine and vinblastine, and more sensitive to colchicine compared to MCF-7CC cells. Live imaging demonstrated that the microtubule dynamics of MCF-7TXT cells were less sensitive to vinca alkaloids, and more sensitive to colchicine. MCF-7TXT cells were also noted to be more sensitive to other CSBAs including 2MeOE2, ABT-751 and phosphorylated combretastatin A-4 (CA-4P)., Conclusion: Docetaxel-resistant MCF-7TXT cells have demonstrated cross-resistance to vinca alkaloids, but appear to be more sensitive to CSBAs (colchicine, 2MeOE2, ABT-751 and CA-4P) compared to non-resistant MCF-7CC cells. Taken together these results suggest that CSBAs should be evaluated further in the treatment of taxane resistant breast cancer.

Background: We previously discovered that tetracyclines increase the expression of lipid phosphate phosphatases at the surface of cells. These enzymes degrade circulating lysophosphatidate and therefore doxycycline increases the turnover of plasma lysophosphatidate and decreases its concentration. Extracellular lysophosphatidate signals through six G protein-coupled receptors and it is a potent promoter of tumor growth, metastasis and chemo-resistance. These effects depend partly on the stimulation of inflammation that lysophosphatidate produces., Methods: In this work, we used a syngeneic orthotopic mouse model of breast cancer to determine the impact of doxycycline on circulating lysophosphatidate concentrations and tumor growth. Cytokine/chemokine concentrations in tumor tissue and plasma were measured by multiplexing laser bead technology. Leukocyte infiltration in tumors was analyzed by immunohistochemistry. The expression of IL-6 in breast cancer cell lines was determined by RT-PCR. Cell growth was measured in Matrigel™ 3D culture. The effects of doxycycline on NF-κB-dependent signaling were analyzed by Western blotting., Results: Doxycycline decreased plasma lysophosphatidate concentrations, delayed tumor growth and decreased the concentrations of several cytokines/chemokines (IL-1β, IL-6, IL-9, CCL2, CCL11, CXCL1, CXCL2, CXCL9, G-CSF, LIF, VEGF) in the tumor. These results were compatible with the effects of doxycycline in decreasing the numbers of F4/80 + macrophages and CD31 + blood vessel endothelial cells in the tumor. Doxycycline also decreased the lysophosphatidate-induced growth of breast cancer cells in three-dimensional culture. Lysophosphatidate-induced Ki-67 expression was inhibited by doxycycline. NF-κB activity in HEK293 cells transiently expressing a NF-κB-luciferase reporter vectors was also inhibited by doxycycline. Treatment of breast cancer cells with doxycycline also decreased the translocation of NF-κB to the nucleus and the mRNA levels for IL-6 in the presence or absence of lysophosphatidate., Conclusion: These results contribute a new dimension for understanding the anti-inflammatory effects of tetracyclines, which make them potential candidates for adjuvant therapy of cancers and other inflammatory diseases.

Recently platelet derived growth factor receptor-alpha (PDGFRα) was recognized as a potential target to treat aggressive papillary thyroid cancer given its strong association with lymph node metastases. However, it is unclear how PDGFRα potentiates metastases and if it works through the canonical MAPK pathway traditionally linked to PTC oncogenesis. We explored the phenotypic changes driven by PDGFRα activation in human papillary thyroid cancer (PTC) cells and the downstream signalling cascades through which they are effected. We demonstrate that PDGFRα drives an impressive phenotypic change in PTC cell lines as documented by significant cytoskeletal rearrangement, increased migratory potential, and the formation of invadopodia. Cells lacking PDGFRα formed compact and dense spheroids, whereas cells expressing active PDGFRα exhibited invadopodia in three-dimensional culture. To achieve this, active PDGFRα provoked downstream activation of the MAPK/Erk, PI3K/Akt and STAT3 pathways. We further confirmed the role of PDGFRα as a transformative agent promoting the epithelial to mesenchymal transition of PTC cells, through the augmentation of Snail and Slug expression. Crenolanib, a small molecule inhibitor of PDGFRα, suppressed the levels of Snail and Slug and almost completely reversed all the phenotypic changes. We demonstrate that PDGFRα activation is an essential component that drives aggressiveness in PTC cells, and that the signaling pathways are complex, involving not only the MAPK/Erk but also the PI3K/Akt and STAT3 pathways. This argues for upstream targeting of the PDGFRα given the redundancy of oncogenic pathways in PTC, especially in patients whose tumors over-express this tyrosine kinase receptor.

Dedifferentiation of follicular cells is a central event in resistance to radioactive iodine and patient mortality in papillary thyroid carcinoma (PTC). We reveal that platelet derived growth factor receptor alpha (PDGFRα) specifically drives dedifferentiation in PTC by disrupting the transcriptional activity of thyroid transcription factor-1 (TTF1). PDGFRα activation dephosphorylates TTF1 consequently shifting the localization of this transcription factor from the nucleus to the cytoplasm. TTF1 is required for follicular cell development and disrupting its function abrogates thyroglobulin production and sodium iodide transport. PDGFRα also promotes a more invasive and migratory cell phenotype with a dramatic increase in xenograft tumor formation. In patient tumors we confirm that nuclear TTF1 expression is inversely proportional to PDGFRα levels. Patients exhibiting PDGFRα at time of diagnosis are three times more likely to exhibit nodal metastases and are 18 times more likely to recur within 5years than those patients lacking PDGFRα expression. Moreover, high levels of PDGFRα and low levels of nuclear TTF1 predict resistance to radioactive iodine therapy. We demonstrate in SCID xenografts that focused PDGFRα blockade restores iodide transport and decreases tumor burden by >50%. Focused PDGFRα inhibitors, combined with radioactive iodine, represent an additional avenue for treating patients with aggressive variants of PTC., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Extracellular lysophosphatidate (LPA) is a potent bioactive lipid that signals through six G-protein-coupled receptors. This signaling is required for embryogenesis, tissue repair and remodeling processes. LPA is produced from circulating lysophosphatidylcholine by autotaxin (ATX), and is degraded outside cells by a family of three enzymes called the lipid phosphate phosphatases (LPPs). In many pathological conditions, particularly in cancers, LPA concentrations are increased due to high ATX expression and low LPP activity. In cancers, LPA signaling drives tumor growth, angiogenesis, metastasis, resistance to chemotherapy and decreased efficacy of radiotherapy. Hence, targeting the ATX-LPA-LPP axis is an attractive strategy for introducing novel adjuvant therapeutic options. In this review, we will summarize current progress in targeting the ATX-LPA-LPP axis with inhibitors of autotaxin activity, LPA receptor antagonists, LPA monoclonal antibodies, and increasing low LPP expression. Some of these agents are already in clinical trials and have applications beyond cancer, including chronic inflammatory diseases., (© 2016 the Journal of Biomedical Research. All rights reserved.)

Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 μg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs., (Copyright © 2016 by the American Society for Biochemistry and Molecular Biology, Inc.)

Tamoxifen is the accepted therapy for patients with estrogen receptor-α (ERα)-positive breast cancer. However, clinical resistance to tamoxifen, as demonstrated by recurrence or progression on therapy, is frequent and precedes death from metastases. To improve breast cancer treatment it is vital to understand the mechanisms that result in tamoxifen resistance. This study shows that concentrations of tamoxifen and its metabolites, which accumulate in tumors of patients, killed both ERα-positive and ERα-negative breast cancer cells. This depended on oxidative damage and anti-oxidants rescued the cancer cells from tamoxifen-induced apoptosis. Breast cancer cells responded to tamoxifen-induced oxidation by increasing Nrf2 expression and subsequent activation of the anti-oxidant response element (ARE). This increased the transcription of anti-oxidant genes and multidrug resistance transporters. As a result, breast cancer cells are able to destroy or export toxic oxidation products leading to increased survival from tamoxifen-induced oxidative damage. These responses in cancer cells also occur in breast tumors of tamoxifen-treated mice. Additionally, high levels of expression of Nrf2, ABCC1, ABCC3 plus NAD(P)H dehydrogenase quinone-1 in breast tumors of patients at the time of diagnosis were prognostic of poor survival after tamoxifen therapy. Therefore, overcoming tamoxifen-induced activation of the ARE could increase the efficacy of tamoxifen in treating breast cancer.

Lipid phosphate phosphatases (LPPs) are a group of enzymes that belong to a phosphatase/phosphotransferase family. Mammalian LPPs consist of three isoforms: LPP1, LPP2, and LPP3. They share highly conserved catalytic domains and catalyze the dephosphorylation of a variety of lipid phosphates, including phosphatidate, lysophosphatidate (LPA), sphingosine 1-phosphate (S1P), ceramide 1-phosphate, and diacylglycerol pyrophosphate. LPPs are integral membrane proteins, which are localized on plasma membranes with the active site on the outer leaflet. This enables the LPPs to degrade extracellular LPA and S1P, thereby attenuating their effects on the activation of surface receptors. LPP3 also exhibits noncatalytic effects at the cell surface. LPP expression on internal membranes, such as endoplasmic reticulum and Golgi, facilitates the metabolism of internal lipid phosphates, presumably on the luminal surface of these organelles. This action probably explains the signaling effects of the LPPs, which occur downstream of receptor activation. The three isoforms of LPPs show distinct and nonredundant effects in several physiological and pathological processes including embryo development, vascular function, and tumor progression. This review is intended to present an up-to-date understanding of the physiological and pathological consequences of changing the activities of the different LPPs, especially in relation to cell signaling by LPA and S1P., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)