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Epoxides and diols of polyunsaturated fatty acids (PUFAs) are bioactive and can influence processes such as tumor cell proliferation and angiogenesis. Studies with inhibitors of the soluble epoxide hydrolase (sEH) in animals overexpressing cytochrome P450 enzymes or following the systemic administration of specific epoxides revealed a markedly increased incidence of tumor metastases. To determine whether PUFA epoxides increased metastases in a model of spontaneous breast cancer, sEH-/- mice were crossed onto the polyoma middle T oncogene (PyMT) background. We found that the deletion of the sEH accelerated the growth of primary tumors and increased both the tumor macrophage count and angiogenesis. There were small differences in the epoxide/diol content of tumors, particularly in epoxyoctadecamonoenic acid versus dihydroxyoctadecenoic acid, and marked changes in the expression of proteins linked with cell proliferation and metabolism. However, there was no consequence of sEH inhibition on the formation of metastases in the lymph node or lung. Taken together, our results confirm previous reports of increased tumor growth in animals lacking sEH but fail to substantiate reports of enhanced lymph node or pulmonary metastases.
In ischemic vascular diseases, leukocyte recruitment and polarization are crucial for revascularization and tissue repair. We investigated the role of vasodilator-stimulated phosphoprotein (VASP) in vascular repair. After hindlimb ischemia induction, blood flow recovery, angiogenesis, arteriogenesis, and leukocyte infiltration into ischemic muscles in VASP−/− mice were accelerated. VASP deficiency also elevated the polarization of the macrophages through increased signal transducer and activator of transcription (STAT) signaling, which augmented the release of chemokines, cytokines, and growth factors to promote leukocyte recruitment and vascular repair. Importantly, VASP deletion in bone marrow–derived cells was sufficient to mimic the increased blood flow recovery of global VASP−/− mice. In chemotaxis experiments, VASP−/− neutrophils/monocytes were significantly more responsive to M1-related chemokines than wild-type controls. Mechanistically, VASP formed complexes with the chemokine receptor CCR2 and β-arrestin-2, and CCR2 receptor internalization was significantly reduced in VASP−/− leukocytes. Our data indicate that VASP is a major regulator of leukocyte recruitment and polarization in postischemic revascularization and support a novel role of VASP in chemokine receptor trafficking.
Hypoxia poses a stress to cells and decreases mitochondrial respiration, in part by electron transport chain (ETC) complex reorganization. While metabolism under acute hypoxia is well characterized, alterations under chronic hypoxia largely remain unexplored. We followed oxygen consumption rates in THP-1 monocytes during acute (16 h) and chronic (72 h) hypoxia, compared to normoxia, to analyze the electron flows associated with glycolysis, glutamine, and fatty acid oxidation. Oxygen consumption under acute hypoxia predominantly demanded pyruvate, while under chronic hypoxia, fatty acid- and glutamine-oxidation dominated. Chronic hypoxia also elevated electron-transferring flavoproteins (ETF), and the knockdown of ETF–ubiquinone oxidoreductase lowered mitochondrial respiration under chronic hypoxia. Metabolomics revealed an increase in citrate under chronic hypoxia, which implied glutamine processing to α-ketoglutarate and citrate. Expression regulation of enzymes involved in this metabolic shunting corroborated this assumption. Moreover, the expression of acetyl-CoA carboxylase 1 increased, thus pointing to fatty acid synthesis under chronic hypoxia. Cells lacking complex I, which experienced a markedly impaired respiration under normoxia, also shifted their metabolism to fatty acid-dependent synthesis and usage. Taken together, we provide evidence that chronic hypoxia fuels the ETC via ETFs, increasing fatty acid production and consumption via the glutamine-citrate-fatty acid axis.
Macrophages exposed to the Th2 cytokines interleukin (IL) IL-4 and IL-13 exhibit a distinct transcriptional response, commonly referred to as M2 polarization. Recently, IL-4-induced polarization of murine bone marrow-derived macrophages (BMDMs) has been linked to acetyl-CoA levels through the activity of the cytosolic acetyl-CoA-generating enzyme ATP-citrate lyase (ACLY). Here, we studied how ACLY regulated IL-4-stimulated gene expression in human monocyte-derived macrophages (MDMs). Although multiple ACLY inhibitors attenuated IL-4-induced target gene expression, this effect could not be recapitulated by silencing ACLY expression. Furthermore, ACLY inhibition failed to alter cellular acetyl-CoA levels and histone acetylation. We generated ACLY knockout human THP-1 macrophages using CRISPR/Cas9 technology. While these cells exhibited reduced histone acetylation levels, IL-4-induced gene expression remained intact. Strikingly, ACLY inhibitors still suppressed induction of target genes by IL-4 in ACLY knockout cells, suggesting off-target effects of these drugs. Our findings suggest that ACLY may not be the major regulator of nucleocytoplasmic acetyl-CoA and IL-4-induced polarization in human macrophages. Furthermore, caution should be warranted in interpreting the impact of pharmacological inhibition of ACLY on gene expression.
MicroRNAs have been projected as promising tools for diagnostic and prognostic purposes in cancer. More recently, they have been highlighted as RNA therapeutic targets for cancer therapy. Though miRs perform a generic function of post-transcriptional gene regulation, their utility in RNA therapeutics mostly relies on their biochemical nature and their assembly with other macromolecules. Release of extracellular miRs is broadly categorized into two different compositions, namely exosomal (extracellular vesicles) and non-exosomal. This nature of miRs not only affects the uptake into target cells but also poses a challenge and opportunity for RNA therapeutics in cancer. By virtue of their ability to act as mediators of intercellular communication in the tumor microenvironment, extracellular miRs perform both, depending upon the target cell and target landscape, pro- and anti-tumor functions. Tumor-derived miRs mostly perform pro-tumor functions, whereas host cell- or stroma-derived miRs are involved in anti-tumor activities. This review deals with the recent understanding of exosomal and non-exosomal miRs in the tumor microenvironment, as a tool for pro- and anti-tumor activity and prospective exploit options for cancer therapy.
Highlights
• TAM polarization induces CP RNA.
• CP RNA expression is regulated by HIF-2 and STAT1.
• CP RNA is transferred from TAMs to HT1080 cells.
• CP RNA is translated by HT1080 cells and protects from ferroptosis.
• Co-cultured HT1080 cells decrease iron and lipid peroxidation.
Abstract
Solid tumors are characterized by hypoxic areas, which are prone for macrophage infiltration. Once infiltrated, macrophages polarize to tumor associated macrophages (TAM) to support tumor progression. Therefore, the crosstalk between TAMs and tumor cells is of current interest for the development of novel therapeutic strategies. These may comprise induction of an iron- and lipid peroxidation-dependent form of cell death, known as ferroptosis. To study the macrophage - tumor cell crosstalk we polarized primary human macrophages towards a TAM-like phenotype, co-cultured them with HT1080 fibrosarcoma cells, and analyzed the tumor cell response to ferroptosis induction. In TAMs the expression of ceruloplasmin mRNA increased, which was driven by hypoxia inducible factor 2 and signal transducer and activator of transcription 1. Subsequently, ceruloplasmin mRNA was transferred from TAMs to HT1080 cells via extracellular vesicles. In tumor cells, mRNA was translated into protein to protect HT1080 cells from RSL3-induced ferroptosis. Mechanistically this was based on reduced iron abundance and lipid peroxidation. Interestingly, in naïve macrophages also hypoxia induced ceruloplasmin under hypoxia and a co-culture of HT1080 cells with hypoxic macrophages recapitulated the protective effect observed in TAM co-cultures. In conclusion, TAMs provoke tumor cells to release iron and thereby protect them from lipid peroxidation/ferroptosis.
Metabolic adaptation and signal integration in response to hypoxic conditions is mainly regulated by hypoxia-inducible factors (HIFs). At the same time, hypoxia induces ROS formation and activates the unfolded protein response (UPR), indicative of endoplasmic reticulum (ER) stress. However, whether ER stress would affect the hypoxia response remains ill-defined. Here we report that feeding mice a high fat diet causes ER stress and attenuates the response to hypoxia. Mechanistically, ER stress promotes HIF-1α and HIF-2α degradation independent of ROS, Ca2+, and the von Hippel-Lindau (VHL) pathway, involving GSK3β and the ubiquitin ligase FBXW1A/βTrCP. Thereby, we reveal a previously unknown function of the GSK3β/HIFα/βTrCP1 axis in ER homeostasis and demonstrate that inhibition of the HIF-1 and HIF-2 response and genetic deficiency of GSK3β affects proliferation, migration, and sensitizes cells for ER stress promoted apoptosis. Vice versa, we show that hypoxia affects the ER stress response mainly through the PERK-arm of the UPR. Overall, we discovered previously unrecognized links between the HIF pathway and the ER stress response and uncovered an essential survival pathway for cells under ER stress.
The sphingolipid sphingosine-1-phosphate (S1P) promotes tumor development through a variety of mechanisms including promoting proliferation, survival, and migration of cancer cells. Moreover, S1P emerged as an important regulator of tumor microenvironmental cell function by modulating, among other mechanisms, tumor angiogenesis. Therefore, S1P was proposed as a target for anti-tumor therapy. The clinical success of current cancer immunotherapy suggests that future anti-tumor therapy needs to consider its impact on the tumor-associated immune system. Hereby, S1P may have divergent effects. On the one hand, S1P gradients control leukocyte trafficking throughout the body, which is clinically exploited to suppress auto-immune reactions. On the other hand, S1P promotes pro-tumor activation of a diverse range of immune cells. In this review, we summarize the current literature describing the role of S1P in tumor-associated immunity, and we discuss strategies for how to target S1P for anti-tumor therapy without causing immune paralysis.
Ferroptosis is an iron-dependent form of cell death, which is triggered by disturbed membrane integrity due to an overproduction of lipid peroxides. Induction of ferroptosis comprises several alterations, i.e. altered iron metabolism, response to oxidative stress, or lipid peroxide production. At the physiological level transcription, translation, and microRNAs add to the appearance and/or activity of building blocks that negatively or positively balance ferroptosis. Ferroptosis contributes to tissue damage in the case of, e.g., brain and heart injury but may be desirable to overcome chemotherapy resistance. For a more complete picture, it is crucial to also consider the cellular microenvironment, which during inflammation and in the tumor context is dominated by hypoxia. This graphical review visualizes basic mechanisms of ferroptosis, categorizes general inducers and inhibitors of ferroptosis, and puts a focus on microRNAs, iron homeostasis, and hypoxia as regulatory components.
The activation of the transcription factor NF-E2-related factor 2 (Nrf2) maintains cellular homeostasis in response to oxidative stress by the regulation of multiple cytoprotective genes. Without stressors, the activity of Nrf2 is inhibited by its interaction with the Keap1 (kelch-like ECH-associated protein 1). Here, we describe (3S)-1-[4-[(2,3,5,6-tetramethylphenyl) sulfonylamino]-1-naphthyl]pyrrolidine-3-carboxylic acid (RA839), a small molecule that binds noncovalently to the Nrf2-interacting kelch domain of Keap1 with a Kd of ∼6 μm, as demonstrated by x-ray co-crystallization and isothermal titration calorimetry. Whole genome DNA arrays showed that at 10 μm RA839 significantly regulated 105 probe sets in bone marrow-derived macrophages. Canonical pathway mapping of these probe sets revealed an activation of pathways linked with Nrf2 signaling. These pathways were also activated after the activation of Nrf2 by the silencing of Keap1 expression. RA839 regulated only two genes in Nrf2 knock-out macrophages. Similar to the activation of Nrf2 by either silencing of Keap1 expression or by the reactive compound 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me), RA839 prevented the induction of both inducible nitric-oxide synthase expression and nitric oxide release in response to lipopolysaccharides in macrophages. In mice, RA839 acutely induced Nrf2 target gene expression in liver. RA839 is a selective inhibitor of the Keap1/Nrf2 interaction and a useful tool compound to study the biology of Nrf2.