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Loss of HIF-1α in macrophages attenuates AhR/ARNT-mediated tumorigenesis in a PAH-driven tumor model
(2016)
Activation of hypoxia-inducible factor (HIF) and macrophage infiltration of solid tumors independently promote tumor progression. As little is known how myeloid HIF affects tumor development, we injected the polycyclic aromatic hydrocarbon (PAH) and procarcinogen 3-methylcholanthrene (MCA; 100 μg/100 μl) subcutaneously into myeloid-specific Hif-1α and Hif-2α knockout mice (C57BL/6J) to induce fibrosarcomas (n = 16). Deletion of Hif-1α but not Hif-2α in macrophages diminished tumor outgrowth in the MCA-model. While analysis of the tumor initiation phase showed comparable inflammation after MCA-injection, metabolism of MCA was impaired in the absence of Hif-1α. An ex vivo macrophage/fibroblast coculture recapitulated reduced DNA damage after MCA-stimulation in fibroblasts of cocultures with Hif-1α LysM-/- macrophages compared to wild type macrophages. A loss of myeloid Hif-1α decreased RNA levels of arylhydrocarbon receptor (AhR)/arylhydrocarbon receptor nuclear translocator (ARNT) targets such as Cyp1a1 because of reduced Arnt but unchanged Ahr expression. Cocultures using Hif-1α LysM-/- macrophages stimulated with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA; 2 μg/ml) also attenuated a DNA damage response in fibroblasts, while the DNA damage-inducing metabolite DMBA-trans-3,4-dihydrodiol remained effective in the absence of Hif-1α. In chemical-induced carcinogenesis, HIF-1α in macrophages maintains ARNT expression to facilitate PAH-biotransformation. This implies a metabolic activation of PAHs in stromal cells, i.e. myeloid-derived cells, to be crucial for tumor initiation.
Hypomethylating agents decitabine and azacytidine are regarded as interchangeable in the treatment of acute myeloid leukemia (AML). However, their mechanisms of action remain incompletely understood, and predictive biomarkers for HMA efficacy are lacking. Here, we show that the bioactive metabolite decitabine triphosphate, but not azacytidine triphosphate, functions as activator and substrate of the triphosphohydrolase SAMHD1 and is subject to SAMHD1-mediated inactivation. Retrospective immunohistochemical analysis of bone marrow specimens from AML patients at diagnosis revealed that SAMHD1 expression in leukemic cells inversely correlates with clinical response to decitabine, but not to azacytidine. SAMHD1 ablation increases the antileukemic activity of decitabine in AML cell lines, primary leukemic blasts, and xenograft models. AML cells acquire resistance to decitabine partly by SAMHD1 up-regulation. Together, our data suggest that SAMHD1 is a biomarker for the stratified use of hypomethylating agents in AML patients and a potential target for the treatment of decitabine-resistant leukemia.
Hepatocellular carcinoma (HCC) is one of the most difficult cancer types to treat. Liver cancer is often diagnosed at late stages and therapeutic treatment is frequently accompanied by development of multidrug resistance. This leads to poor outcomes for cancer patients. Understanding the fundamental molecular mechanisms leading to liver cancer development is crucial for developing new therapeutic approaches, which are more efficient in treating cancer. Mice with a liver specific UDP-glucose ceramide glucosyltransferase (UGCG) knockout (KO) show delayed diethylnitrosamine (DEN)-induced liver tumor growth. Accordingly, the rationale for our study was to determine whether UGCG overexpression is sufficient to drive cancer phenotypes in liver cells. We investigated the effect of UGCG overexpression (OE) on normal murine liver (NMuLi) cells. Increased UGCG expression results in decreased mitochondrial respiration and glycolysis, which is reversible by treatment with EtDO-P4, an UGCG inhibitor. Furthermore, tumor markers such as FGF21 and EPCAM are lowered following UGCG OE, which could be related to glucosylceramide (GlcCer) and lactosylceramide (LacCer) accumulation in glycosphingolipid-enriched microdomains (GEMs) and subsequently altered signaling protein phosphorylation. These cellular processes lead to decreased proliferation in NMuLi/UGCG OE cells. Our data show that increased UGCG expression itself does not induce pro-cancerous processes in normal liver cells, which indicates that increased GlcCer expression leads to different outcomes in different cancer types.
Based on increasing evidence suggesting that MS pathology involves alterations in bioactive lipid metabolism, the present analysis was aimed at generating a complex serum lipid-biomarker. Using unsupervised machine-learning, implemented as emergent self-organizing maps of neuronal networks, swarm intelligence and Minimum Curvilinear Embedding, a cluster structure was found in the input data space comprising serum concentrations of d = 43 different lipid-markers of various classes. The structure coincided largely with the clinical diagnosis, indicating that the data provide a basis for the creation of a biomarker (classifier). This was subsequently assessed using supervised machine-learning, implemented as random forests and computed ABC analysis-based feature selection. Bayesian statistics-based biomarker creation was used to map the diagnostic classes of either MS patients (n = 102) or healthy subjects (n = 301). Eight lipid-markers passed the feature selection and comprised GluCerC16, LPA20:4, HETE15S, LacCerC24:1, C16Sphinganine, biopterin and the endocannabinoids PEA and OEA. A complex classifier or biomarker was developed that predicted MS at a sensitivity, specificity and accuracy of approximately 95% in training and test data sets, respectively. The present successful application of serum lipid marker concentrations to MS data is encouraging for further efforts to establish an MS biomarker based on serum lipidomics.
Background: Caloric restriction is associated with broad therapeutic potential in various diseases and an increase in health and life span. In this study, we assessed the impact of caloric restriction on acute and inflammatory nociception in mice, which were either fed ad libitum or subjected to caloric restriction with 80% of the daily average for two weeks.
Results: The behavioral tests revealed that inflammatory nociception in the formalin test and in zymosan-induced mechanical hypersensitivity were significantly decreased when mice underwent caloric restriction. As potential mediators of the diet-induced antinociception, we assessed genes typically induced by inflammatory stimuli, AMP-activated kinase, and the endocannabinoid system which have all already been associated with nociceptive responses. Zymosan-induced inflammatory markers such as COX-2, TNFα, IL-1β, and c-fos in the spinal cord were not altered by caloric restriction. In contrast, AMPKα2 knock-out mice showed significant differences in comparison to C57BL/6 mice and their respective wild type littermates by missing the antinociceptive effects after caloric restriction. Endocannabinoid levels of anandamide and 2-arachidonyl glyceroldetermined in serum by LC-MS/MS were not affected by either caloric restriction alone or in combination with zymosan treatment. However, cannabinoid receptor type 1 expression in the spinal cord, which was not altered by caloric restriction in control mice, was significantly increased after caloric restriction in zymosan-induced paw inflammation. Since increased cannabinoid receptor type 1 signaling might influence AMP-activated kinase activity, we analyzed effects of anandamide on AMP-activated kinase in cell culture and observed a significant activation of AMP-activated kinase. Thus, endocannabionoid-induced AMP-activated kinase activation might be involved in antinociceptive effects after caloric restriction.
Conclusion: Our data suggest that caloric restriction has an impact on inflammatory nociception which might involve AMP-activated kinase activation and an increased activity of the endogenous endocannabinoid system by caloric restriction-induced cannabinoid receptor type 1 upregulation.
The activation and infiltration of polymorphonuclear neutrophils (PMN) are critical key steps in inflammation. PMN-mediated inflammation is limited by anti-inflammatory and pro-resolving mechanisms, including specialized pro-resolving lipid mediators (SPM). We examined the effects of 15-epi-LXA4 on inflammation and the biosynthesis of pro-inflammatory mediators, such as prostaglandins, leukotriene B4 and various hydroxyeicosatetraenoic acids and SPM, in an oxazolone (OXA)-induced hypersensitivity model for dermal inflammation. 15-epi-LXA4 (100 μM, 5 μL subcutaneously injected) significantly (P < 0.05) reduced inflammation in skin, 24 hours after the OXA challenge, as compared to skin treated with vehicle. No significant influence on the biosynthesis of prostaglandins or leukotriene B4 was observed, whereas the level of 15S-hydroxy-eicosatetraenoic acid was significantly (P < 0.05) lower in the skin areas treated with 15-epi-LXA4. In spite of the use of a fully validated analytical procedure, no SPM were detected in the biological samples. To investigate the reason for the lack of analytical signal, we tried to mimic the production of SPM (lipoxins, resolvins, maresin and protectin) by injecting them subcutaneously into the skin of mice and studying the in vivo availability and distribution of the compounds. All analytes showed very little lateral distribution in skin tissue and their levels were markedly decreased (> 95%) 2 hours after injection. However, docosahexaenoic acid derivatives were biologically more stable than SPM derived from arachidonic acid or eicosapentaenoic acid.
Cancer-induced pain occurs frequently in patients when tumors or their metastases grow in the proximity of nerves. Although this cancer-induced pain states poses an important therapeutical problem, the underlying pathomechanisms are not understood. Here, we implanted adenocarcinoma, fibrosarcoma and melanoma tumor cells in proximity of the sciatic nerve. All three tumor types caused mechanical hypersensitivity, thermal hyposensitivity and neuronal damage. Surprisingly the onset of the hypersensitivity was independent of physical contact of the nerve with the tumors and did not depend on infiltration of cancer cells in the sciatic nerve. However, macrophages and dendritic cells appeared on the outside of the sciatic nerves with the onset of the hypersensitivity. At the same time point downregulation of perineural tight junction proteins was observed, which was later followed by the appearance of microlesions. Fitting to the changes in the epi-/perineurium, a dramatic decrease of triglycerides and acylcarnitines in the sciatic nerves as well as an altered localization and appearance of epineural adipocytes was seen. In summary, the data show an inflammation at the sciatic nerves as well as an increased perineural and epineural permeability. Thus, interventions aiming to suppress inflammatory processes at the sciatic nerve or preserving peri- and epineural integrity may present new approaches for the treatment of tumor-induced pain.
Macrophages are highly versatile cells, which acquire, depending on their microenvironment, pro- (M1-like), or antiinflammatory (M2-like) phenotypes. Here, we studied the role of the G-protein coupled receptor G2A (GPR132), in chemotactic migration and polarization of macrophages, using the zymosan-model of acute inflammation. G2A-deficient mice showed a reduced zymosan-induced thermal hyperalgesia, which was reversed after macrophage depletion. Fittingly, the number of M1-like macrophages was reduced in the inflamed tissue in G2A-deficient mice. However, G2A activation was not sufficient to promote M1-polarization in bone marrow-derived macrophages. While the number of monocyte-derived macrophages in the inflamed paw was not altered, G2A-deficient mice had less macrophages in the direct vicinity of the origin of inflammation, an area marked by the presence of zymosan, neutrophil accumulation and proinflammatory cytokines. Fittingly neutrophil efferocytosis was decreased in G2A-deficient mice and several lipids, which are released by neutrophils and promote G2A-mediated chemotaxis, were increased in the inflamed tissue. Taken together, G2A is necessary to position macrophages in the proinflammatory microenvironment surrounding the center of inflammation. In absence of G2A the macrophages are localized in an antiinflammatory microenvironment and macrophage polarization is shifted toward M2-like macrophages.
Dysregulation of lysophosphatidic acids in multiple sclerosis and autoimmune encephalomyelitis
(2017)
Bioactive lipids contribute to the pathophysiology of multiple sclerosis. Here, we show that lysophosphatidic acids (LPAs) are dysregulated in multiple sclerosis (MS) and are functionally relevant in this disease. LPAs and autotaxin, the major enzyme producing extracellular LPAs, were analyzed in serum and cerebrospinal fluid in a cross-sectional population of MS patients and were compared with respective data from mice in the experimental autoimmune encephalomyelitis (EAE) model, spontaneous EAE in TCR1640 mice, and EAE in Lpar2 -/- mice. Serum LPAs were reduced in MS and EAE whereas spinal cord LPAs in TCR1640 mice increased during the ‘symptom-free’ intervals, i.e. on resolution of inflammation during recovery hence possibly pointing to positive effects of brain LPAs during remyelination as suggested in previous studies. Peripheral LPAs mildly re-raised during relapses but further dropped in refractory relapses. The peripheral loss led to a redistribution of immune cells from the spleen to the spinal cord, suggesting defects of lymphocyte homing. In support, LPAR2 positive T-cells were reduced in EAE and the disease was intensified in Lpar2 deficient mice. Further, treatment with an LPAR2 agonist reduced clinical signs of relapsing-remitting EAE suggesting that the LPAR2 agonist partially compensated the endogenous loss of LPAs and implicating LPA signaling as a novel treatment approach.
Persistent and, in particular, neuropathic pain is a major healthcare problem with still insufficient pharmacological treatment options. This triggered research activities aimed at finding analgesics with a novel mechanism of action. Results of these efforts will need to pass through the phases of drug development, in which experimental human pain models are established components e.g. implemented as chemical hyperalgesia induced by capsaicin. We aimed at ranking the various readouts of a human capsaicin–based pain model with respect to the most relevant information about the effects of a potential reference analgesic. In a placebo‐controlled, randomized cross‐over study, seven different pain‐related readouts were acquired in 16 healthy individuals before and after oral administration of 300 mg pregabalin. The sizes of the effect on pain induced by intradermal injection of capsaicin were quantified by calculating Cohen's d. While in four of the seven pain‐related parameters, pregabalin provided a small effect judged by values of Cohen's d exceeding 0.2, an item categorization technique implemented as computed ABC analysis identified the pain intensities in the area of secondary hyperalgesia and of allodynia as the most suitable parameters to quantify the analgesic effects of pregabalin. Results of this study provide further support for the ability of the intradermal capsaicin pain model to show analgesic effects of pregabalin. Results can serve as a basis for the designs of studies where the inclusion of this particular pain model and pregabalin is planned.