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Previous studies towards reduced oxygen availability have mostly focused on changes in total mRNA expression, neglecting underlying transcriptional and post-transcriptional events. Therefore, we generated a comprehensive overview of hypoxia-induced changes in total mRNA expression, global de novo transcription, and mRNA stability in monocytic THP-1 cells. Since hypoxic episodes often persist for prolonged periods, we further compared the adaptation to acute and chronic hypoxia. While total mRNA changes correlated well with enhanced transcription during short-term hypoxia, mRNA destabilization gained importance under chronic conditions. Reduced mRNA stability not only added to a compensatory attenuation of immune responses, but also, most notably, to the reduction in nuclear-encoded mRNAs associated with various mitochondrial functions. These changes may prevent the futile production of new mitochondria under conditions where mitochondria cannot exert their full metabolic function and are indeed actively removed by mitophagy. The post-transcriptional mode of regulation might further allow for the rapid recovery of mitochondrial capacities upon reoxygenation. Our results provide a comprehensive resource of functional mRNA expression dynamics and underlying transcriptional and post-transcriptional regulatory principles during the adaptation to hypoxia. Furthermore, we uncover that RNA stability regulation controls mitochondrial functions in the context of hypoxia.
While the importance of the iron-load of lipocalin-2 (Lcn-2) in promoting tumor progression is widely appreciated, underlying molecular mechanisms largely remain elusive. Considering its role as an iron-transporter, we aimed at clarifying iron-loaded, holo-Lcn-2 (hLcn-2)-dependent signaling pathways in affecting renal cancer cell viability. Applying RNA sequencing analysis in renal CAKI1 tumor cells to explore highly upregulated molecular signatures in response to hLcn-2, we identified a cluster of genes (SLC7A11, GCLM, GLS), which are implicated in regulating ferroptosis. Indeed, hLcn-2-stimulated cells are protected from erastin-induced ferroptosis. We also noticed a rapid increase in reactive oxygen species (ROS) with subsequent activation of the antioxidant Nrf2 pathway. However, knocking down Nrf2 by siRNA was not sufficient to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. In contrast, preventing oxidative stress through N-acetyl-l-cysteine (NAC) supplementation was still able to induce erastin-dependent ferroptotic cell death in hLcn-2-stimulated tumor cells. Besides an oxidative stress response, we noticed activation of the integrated stress response (ISR), shown by enhanced phosphorylation of eIF-2α and induction of ATF4 after hLcn-2 addition. ATF4 knockdown as well as inhibition of the ISR sensitized hLcn-2-treated renal tumor cells to ferroptosis, thus linking the ISR to pro-tumor characteristics of hLcn-2. Our study provides mechanistic details to better understand tumor pro-survival pathways initiated by iron-loaded Lcn-2.
The nuclear factor kappa beta (NFκB) signaling pathway plays an important role in liver homeostasis and cancer development. Tax1-binding protein 1 (Tax1BP1) is a regulator of the NFκB signaling pathway, but its role in the liver and hepatocellular carcinoma (HCC) is presently unknown. Here we investigated the role of Tax1BP1 in liver cells and murine models of HCC and liver fibrosis. We applied the diethylnitrosamine (DEN) model of experimental hepatocarcinogenesis in Tax1BP1+/+ and Tax1BP1−/− mice. The amount and subsets of non-parenchymal liver cells in in Tax1BP1+/+ and Tax1BP1−/− mice were determined and activation of NFκB and stress induced signaling pathways were assessed. Differential expression of mRNA and miRNA was determined. Tax1BP1−/− mice showed increased numbers of inflammatory cells in the liver. Furthermore, a sustained activation of the NFκB signaling pathway was found in hepatocytes as well as increased transcription of proinflammatory cytokines in isolated Kupffer cells from Tax1BP1−/− mice. Several differentially expressed mRNAs and miRNAs in livers of Tax1BP1−/− mice were found, which are regulators of inflammation or are involved in cancer development or progression. Furthermore, Tax1BP1−/− mice developed more HCCs than their Tax1BP1+/+ littermates. We conclude that Tax1BP1 protects from liver cancer development by limiting proinflammatory signaling.
The tumor-microenvironment (TME) is an amalgamation of various factors derived from malignant cells and infiltrating host cells, including cells of the immune system. One of the important factors of the TME is microRNAs (miRs) that regulate target gene expression at a post transcriptional level. MiRs have been found to be dysregulated in tumor as well as in stromal cells and they emerged as important regulators of tumorigenesis. In fact, miRs regulate almost all hallmarks of cancer, thus making them attractive tools and targets for novel anti-tumoral treatment strategies. Tumor to stroma cell cross-propagation of miRs to regulate protumoral functions has been a salient feature of the TME. MiRs can either act as tumor suppressors or oncogenes (oncomiRs) and both miR mimics as well as miR inhibitors (antimiRs) have been used in preclinical trials to alter cancer and stromal cell phenotypes. Owing to their cascading ability to regulate upstream target genes and their chemical nature, which allows specific pharmacological targeting, miRs are attractive targets for anti-tumor therapy. In this review, we cover a recent update on our understanding of dysregulated miRs in the TME and provide an overview of how these miRs are involved in current cancer-therapeutic approaches from bench to bedside.
MicroRNAs (miRs) significantly contribute to the regulation of gene expression, by virtue of their ability to interact with a broad, yet specific set of target genes. MiRs are produced and released by almost every cell type and play an important role in horizontal gene regulation in the tumor microenvironment (TME). In the TME, both tumor and stroma cells cross-communicate via diverse factors including miRs, which are taking central stage as a therapeutic target of anti-tumor therapy. One of the immune escape strategies adopted by tumor cells is to release miRs as a Trojan horse to hijack circulating or tumor-localized monocytes/macrophages to tune them for pro-tumoral functions. On the other hand, macrophage-derived miRs exert anti-tumor functions. The transfer of miRs from host to recipient cells depends on the supramolecular structure and composition of miR carriers, which determine the distinct uptake mechanism by recipient cells. In this review, we provide a recent update on the miR-mediated crosstalk between tumor cells and macrophages and their mode of uptake in the TME.
A myriad of signaling molecules in a heuristic network of the tumor microenvironment (TME) pose a challenge and an opportunity for novel therapeutic target identification in human cancers. MicroRNAs (miRs), due to their ability to affect signaling pathways at various levels, take a prominent space in the quest of novel cancer therapeutics. The role of miRs in cancer initiation, progression, as well as in chemoresistance, is being increasingly investigated. The canonical function of miRs is to target mRNAs for post-transcriptional gene silencing, which has a great implication in first-order regulation of signaling pathways. However, several reports suggest that miRs also perform non-canonical functions, partly due to their characteristic non-coding small RNA nature. Examples emerge when they act as ligands for toll-like receptors or perform second-order functions, e.g., to regulate protein translation and interactions. This review is a compendium of recent advancements in understanding the role of miRs in cancer signaling and focuses on the role of miRs as novel regulators of the signaling pathway in the TME.
Recent studies suggested an important contribution of sphingosine-1-phospate (S1P) signaling via its specific receptors (S1PRs) in the production of pro-inflammatory mediators such as Interleukin (IL)-1β in cancer and inflammation. In an inflammation-driven cancer setting, we previously reported that myeloid S1PR1 signaling induces IL-1β production by enhancing NLRP3 (NOD-, LRR- and Pyrin Domain-Containing Protein 3) inflammasome activity. However, the autocrine role of S1P and enzymes acting on the S1P rheostat in myeloid cells are unknown. Using human and mouse macrophages with pharmacological or genetic intervention we explored the relative contribution of sphingosine kinases (SPHKs) in NLRP3 inflammasome activity regulation. We noticed redundancy in SPHK1 and SPHK2 activities towards macrophage NLRP3 inflammasome transcriptional induction and IL-1β secretion. However, pharmacological blockade of both kinases in unison completely abrogated NLRP3 inflammasome induction and IL-1β secretion. Interestingly, human and mouse macrophages demonstrate varied responses towards SPHKs inhibition and IL-1β secretion. Clinical datasets of renal cell carcinoma and psoriasis patients showed a positive correlation between enzymes affecting the S1P rheostat with NLRP3 inflammasome components expression, which corroborates our finding. Our data provide a better understanding on the role of SPHKs and de novo synthesized S1P in macrophage NLRP3 inflammasome activation
Macrophages supply iron to the breast tumor microenvironment by enforced secretion of lipocalin-2 (Lcn-2)-bound iron as well as the increased expression of the iron exporter ferroportin (FPN). We aimed at identifying the contribution of each pathway in supplying iron for the growing tumor, thereby fostering tumor progression. Analyzing the expression profiles of Lcn-2 and FPN using the spontaneous polyoma-middle-T oncogene (PyMT) breast cancer model as well as mining publicly available TCGA (The Cancer Genome Atlas) and GEO Series(GSE) datasets from the Gene Expression Omnibus database (GEO), we found no association between tumor parameters and Lcn-2 or FPN. However, stromal/macrophage-expression of Lcn-2 correlated with tumor onset, lung metastases, and recurrence, whereas FPN did not. While the total iron amount in wildtype and Lcn-2−/− PyMT tumors showed no difference, we observed that tumor-associated macrophages from Lcn-2−/− compared to wildtype tumors stored more iron. In contrast, Lcn-2−/− tumor cells accumulated less iron than their wildtype counterparts, translating into a low migratory and proliferative capacity of Lcn-2−/− tumor cells in a 3D tumor spheroid model in vitro. Our data suggest a pivotal role of Lcn-2 in tumor iron-management, affecting tumor growth. This study underscores the role of iron for tumor progression and the need for a better understanding of iron-targeted therapy approaches.
Mitochondrial derived reactive oxygen species (mtROS) are known for their signaling qualities in both physiology and pathology. To elucidate mitochondrial complex I-dependent ROS-signaling after lipopolysaccharide (LPS)-stimulation THP-1 macrophages with a knockdown of the transmembrane protein TMEM126B were generated. TMEM knockdown cells (sh126B) showed a reduced assembly of complex I and attenuated mtROS production. In these cells we identified protein oxidization by mtROS upon LPS-treatment using the BIAM switch assay coupled to liquid chromatography and mass spectrometry. One of the identified targets of mtROS was succinate dehydrogenase (SDH) flavoprotein subunit A (SDHA). Oxidation of SDHA decreased its enzymatic activity and pharmacological inhibition of SDH in turn stabilized hypoxia inducible factor (HIF)-1α and caused the subsequent, sustained expression of interleukin-1β (IL-1β). Oxidation of SDHA in sh126B cells was attenuated, while pharmacological inhibition of SDH by atpenin A5 restored IL-1β expression in sh126B cells upon LPS-treatment. Conclusively, oxidation of SDH by mtROS links an altered metabolism, i.e. succinate accumulation to HIF-1-driven, inflammatory changes in macrophages.
Sepsis is characterized by dysregulated gene expression, provoking a hyper-inflammatory response occurring in parallel to a hypo-inflammatory reaction. This is often associated with multi-organ failure, leading to the patient’s death. Therefore, reprogramming of these pro- and anti-inflammatory, as well as immune-response genes which are involved in acute systemic inflammation, is a therapy approach to prevent organ failure and to improve sepsis outcomes. Considering epigenetic, i.e., reversible, modifications of chromatin, not altering the DNA sequence as one tool to adapt the expression profile, inhibition of factors mediating these changes is important. Acetylation of histones by histone acetyltransferases (HATs) and initiating an open-chromatin structure leading to its active transcription is counteracted by histone deacetylases (HDACs). Histone deacetylation triggers a compact nucleosome structure preventing active transcription. Hence, inhibiting the activity of HDACs by specific inhibitors can be used to restore the expression profile of the cells. It can be assumed that HDAC inhibitors will reduce the expression of pro-, as well as anti-inflammatory mediators, which blocks sepsis progression. However, decreased cytokine expression might also be unfavorable, because it can be associated with decreased bacterial clearance.
Carcinogenesis is a multistep process. Besides somatic mutations in tumor cells, stroma-associated immunity is a major regulator of tumor growth. Tumor cells produce and secrete diverse mediators to create a local microenvironment that supports their own survival and growth. It is becoming apparent that iron acquisition, storage, and release in tumor cells is different from healthy counterparts. It is also appreciated that macrophages in the tumor microenvironment acquire a tumor-supportive, anti-inflammatory phenotype that promotes tumor cell proliferation, angiogenesis, and metastasis. Apparently, this behavior is attributed, at least in part, to the ability of macrophages to support tumor cells with iron. Polarization of macrophages by apoptotic tumor cells shifts the profile of genes involved in iron metabolism from an iron sequestering to an iron-release phenotype. Iron release from macrophages is supposed to be facilitated by ferroportin. However, lipid mediators such as sphingosine-1-phosphate, released form apoptotic tumor cells, upregulate lipocalin-2 (Lcn-2) in macrophages. This protein is known to bind siderophore-complexed iron and thus, may participate in iron transport in the tumor microenvironment. We describe how macrophages handle iron in the tumor microenvironment, discuss the relevance of an iron-release macrophage phenotype for tumor progression, and propose a new role for Lcn-2 in tumor-associated macrophages.
Accumulating evidence suggests that iron homeostasis is disturbed in tumors. We aimed at clarifying the distribution of iron in renal cell carcinoma (RCC). Considering the pivotal role of macrophages for iron homeostasis and their association with poor clinical outcome, we investigated the role of macrophage-secreted iron for tumor progression by applying a novel chelation approach. We applied flow cytometry and multiplex-immunohistochemistry to detect iron-dependent markers and analyzed iron distribution with atomic absorption spectrometry in patients diagnosed with RCC. We further analyzed the functional significance of iron by applying a novel extracellular chelator using RCC cell lines as well as patient-derived primary cells. The expression of iron-regulated genes was significantly elevated in tumors compared to adjacent healthy tissue. Iron retention was detected in tumor cells, whereas tumor-associated macrophages showed an iron-release phenotype accompanied by enhanced expression of ferroportin. We found increased iron amounts in extracellular fluids, which in turn stimulated tumor cell proliferation and migration. In vitro, macrophage-derived iron showed pro-tumor functions, whereas application of an extracellular chelator blocked these effects. Our study provides new insights in iron distribution and iron-handling in RCC. Chelators that specifically scavenge iron in the extracellular space confirmed the importance of macrophage-secreted iron in promoting tumor growth
Background: The ligand-activated transcription factor, peroxisome-proliferator-activated receptor gamma (PPARγ), has been shown to play an essential role in immunosuppression during sepsis. PPARγ is upregulated in T cells of septic patients, sensitizing these cells to PPARγ-dependent apoptosis and thus contributing to T-cell depletion. In the polymicrobial cecum ligation and puncture (CLP) sepsis model in mice, both T-cell-specific gene knockout (Lck-Cre PPARγfl/fl) and systemic pharmacological PPARγ antagonism by GW9662 improved survival. Because GW9662 was only effective when applied 3 hours after CLP, we were interested to extend this time frame. For this reason we characterized the kinetics of SPPARγMs when administered before or in combination with the agonist thiazolidinedione, rosiglitazone.
Methods: A PPARγ-dependent transactivation assay was used in HEK293T cells. It is based on the vector pFA-PPARγ-LBD-GAL4-DBD encoding the hybrid protein PPARγ-LBD-GAL4-DBD and the reporter vector pFR-Luc, carrying a GAL4-responsive element in front of the Firefly luciferase gene. These two vectors were co-transfected, in combination with a control vector encoding Renilla luciferase (pRL-CMV) to normalize Firefly luciferase activity for transfection efficiency. Following transfection, cells were incubated with the SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 for different times (2 to 48 hours) and at increasing doses (0.01 to 10 μM), with or without rosiglitazone (0.01 to 10 μM). Transactivation was analyzed using a 96-well plate format.
Results: Rosiglitazone transactivated PPARγ in a time-dependent and dose-dependent manner, the response gradually increasing to a maximum at 48 hours with 10 μM. Low concentrations (0.01 to 0.1 μM) of SPPARγMs F-MOC and MCC-555 and the PPARγ antagonist GW9662 all exerted dose-independent antagonistic effects at an early incubation time point (2 hours). From 10 hours onwards, MCC-555 and GW9662, given alone, both exerted PPARγ agonistic effects, MCC-555 in parallel to responses to rosiglitazone, but GW9662 with characteristics of partial antagonism. F-MOC showed no dose-dependent effect at any concentration at later time points. Only GW9662 (1 to 10 μM) was able to inhibit rosiglitazone (0.1 to 1 μM)-induced PPARγ transactivation after 10 hours.
Conclusion: Our kinetic analysis reveals clear differences in the modulatory characteristics of PPARγ inhibitors, with previously unreported early inhibitory effects and late agonistic or partial agonistic activity. New SPPARγMs with extended inhibitory activity may prove useful in the therapy of sepsis.
Tight regulation of inflammation is very important to guarantee a balanced immune response without developing chronic inflammation. One of the major mediators of the resolution of inflammation is the transcription factor: the nuclear factor erythroid 2-like 2 (Nrf2). Stabilized following oxidative stress, Nrf2 induces the expression of antioxidants as well as cytoprotective genes, which provoke an anti-inflammatory expression profile, and is crucial for the initiation of healing. In view of this fundamental modulatory role, it is clear that both hyper- or hypoactivation of Nrf2 contribute to the onset of chronic diseases. Understanding the tight regulation of Nrf2 expression/activation and its interaction with signaling pathways, known to affect inflammatory processes, will facilitate development of therapeutic approaches to prevent Nrf2 dysregulation and ameliorate chronic inflammatory diseases. We discuss in this review the principle mechanisms of Nrf2 regulation with a focus on inflammation and autophagy, extending the role of dysregulated Nrf2 to chronic diseases and tumor development.
Human macrophages infiltrating hypoxic regions alter their metabolism, because oxygen becomes limited. Increased glycolysis is one of the most common cellular adaptations to hypoxia and mostly is regulated via hypoxia-inducible factor (HIF) and RAC-alpha serine/threonine–protein kinase (Akt) signaling, which gets activated under reduced oxygen content. We noticed that micro RNA (miR)-193a-3p enhances Akt phosphorylation at threonine 308 under hypoxia. In detail, miR-193a-3p suppresses the protein abundance of phosphatase PTC7 homolog (PPTC7), which in turn increases Akt phosphorylation. Lowering PPTC7 expression by siRNA or overexpressing miR-193a-3p increases Akt phosphorylation. Vice versa, inhibition of miR-193a-3p attenuates Akt activation and prevents a subsequent increase of glycolysis under hypoxia. Excluding effects of miR-193a-3p and Akt on HIF expression, stabilization, and function, we noticed phosphorylation of 6 phosphofructo-2-kinase/fructose 2,6-bisphosphatase PFKFB3 in response to the PI3K/Akt/mTOR signaling cascade. Inhibition of PFKFB3 blocked an increased glycolytic flux under hypoxia. Apparently, miR-193a-3p balances Akt phosphorylation and dephosphorylation by affecting PPTC7 protein amount. Suppression of PPTC7 increases Akt activation and phosphorylation of PFKFB3, which culminates in higher rates of glycolysis under hypoxia.
The translation inhibitor and tumor suppressor Pdcd4 was reported to be lost in various tumors and put forward as prognostic marker in tumorigenesis. Decreased Pdcd4 protein stability due to PI3K-mTOR-p70S6K1 dependent phosphorylation of Pdcd4 followed by β-TrCP1-mediated ubiquitination, and proteasomal destruction of the protein was characterized as a major mechanism contributing to the loss of Pdcd4 expression in tumors. In an attempt to identify stabilizers of Pdcd4, we used a luciferase-based high-throughput compatible cellular assay to monitor phosphorylation-dependent proteasomal degradation of Pdcd4 in response to mitogen stimulation. Following a screen of approximately 2000 compounds, we identified 1,2-bis(4-chlorophenyl)disulfide as a novel Pdcd4 stabilizer. To determine an initial structure-activity relationship, we used 3 additional compounds, synthesized according to previous reports, and 2 commercially available compounds for further testing, in which either the linker between the aryls was modified (compounds 2–4) or the chlorine residues were replaced by groups with different electronic properties (compounds 5 and 6). We observed that those compounds with alterations in the sulfide linker completely lost the Pdcd4 stabilizing potential. In contrast, modifications in the chlorine residues showed only minor effects on the Pdcd4 stabilizing activity. A reporter with a mutated phospho-degron verified the specificity of the compounds for stabilizing the Pdcd4 reporter. Interestingly, the active diaryl disulfides inhibited proliferation and viability at concentrations where they stabilized Pdcd4, suggesting that Pdcd4 stabilization might contribute to the anti-proliferative properties. Finally, computational modelling indicated that the flexibility of the disulfide linker might be necessary to exert the biological functions of the compounds, as the inactive compound appeared to be energetically more restricted.
While aberrant cells are routinely recognized and removed by immune cells, tumors eventually escape innate immune responses. Infiltrating immune cells are even corrupted by the tumor to acquire a tumor-supporting phenotype. In line, tumor-associated macrophages are well-characterized to promote tumor progression and high levels of tumor-infiltrating macrophages are a poor prognostic marker in breast cancer. Here, we aimed to further decipher the influence of macrophages on breast tumor cells and determined global gene expression changes in three-dimensional tumor spheroids upon infiltration of macrophages. While various tumor-associated mRNAs were upregulated, expression of the cytochrome P450 family member CYP1A1 was markedly attenuated. Repression of CYP1A1 in tumor cells was elicited by a macrophage-shaped tumor microenvironment rather than by direct tumor cell-macrophage contacts. In line with changes in RNA expression profiles, macrophages enhanced proliferation of the tumor cells. Enhanced proliferation and macrophage presence further correlated with reduced CYP1A1 expression in patient tumors when compared with normal tissue. These findings are of interest in the context of combinatory therapeutic approaches involving cytotoxic and immune-modulatory compounds.
Inflammation is a crucial part of immune responses towards invading pathogens or tissue damage. While inflammatory reactions are aimed at removing the triggering stimulus, it is important that these processes are terminated in a coordinate manner to prevent excessive tissue damage due to the highly reactive inflammatory environment. Initiation of inflammatory responses was proposed to be regulated predominantly at a transcriptional level, whereas post-transcriptional modes of regulation appear to be crucial for resolution of inflammation. The RNA-binding protein tristetraprolin (TTP) interacts with AU-rich elements in the 3′ untranslated region of mRNAs, recruits deadenylase complexes and thereby facilitates degradation of its targets. As TTP regulates the mRNA stability of numerous inflammatory mediators, it was put forward as a crucial post-transcriptional regulator of inflammation. Here, we summarize the current understanding of the function of TTP with a specific focus on its role in adding to resolution of inflammation.
Rapid alterations in protein expression are commonly regulated by adjusting translation. In addition to cap-dependent translation, which is e.g. induced by pro-proliferative signaling via the mammalian target of rapamycin (mTOR)-kinase, alternative modes of translation, such as internal ribosome entry site (IRES)-dependent translation, are often enhanced under stress conditions, even if cap-dependent translation is attenuated. Common stress stimuli comprise nutrient deprivation, hypoxia, but also inflammatory signals supplied by infiltrating immune cells. Yet, the impact of inflammatory microenvironments on translation in tumor cells still remains largely elusive. In the present study, we aimed at identifying translationally deregulated targets in tumor cells under inflammatory conditions. Using polysome profiling and microarray analysis, we identified cyp24a1 (1,25-dihydroxyvitamin D3 24-hydroxylase) to be translationally upregulated in breast tumor cells co-cultured with conditioned medium of activated monocyte-derived macrophages (CM). Using bicistronic reporter assays, we identified and validated an IRES within the 5′ untranslated region (5′UTR) of cyp24a1, which enhances translation of cyp24a1 upon CM treatment. Furthermore, IRES-dependent translation of cyp24a1 by CM was sensitive to phosphatidyl-inositol-3-kinase (PI3K) inhibition, while constitutive activation of Akt sufficed to induce its IRES activity. Our data provide evidence that cyp24a1 expression is translationally regulated via an IRES element, which is responsive to an inflammatory environment. Considering the negative feedback impact of cyp24a1 on the vitamin D responses, the identification of a novel, translational mechanism of cyp24a1 regulation might open new possibilities to overcome the current limitations of vitamin D as tumor therapeutic option.
Cancer-associated fibroblasts (CAFs) in the tumor microenvironment contribute to all stages of tumorigenesis and are usually considered to be tumor-promoting cells. CAFs show a remarkable degree of heterogeneity, which is attributed to developmental origin or to local environmental niches, resulting in distinct CAF subsets within individual tumors. While CAF heterogeneity is frequently investigated in late-stage tumors, data on longitudinal CAF development in tumors are lacking. To this end, we used the transgenic polyoma middle T oncogene-induced mouse mammary carcinoma model and performed whole transcriptome analysis in FACS-sorted fibroblasts from early- and late-stage tumors. We observed a shift in fibroblast populations over time towards a subset previously shown to negatively correlate with patient survival, which was confirmed by multispectral immunofluorescence analysis. Moreover, we identified a transcriptomic signature distinguishing CAFs from early- and late-stage tumors. Importantly, the signature of early-stage CAFs correlated well with tumor stage and survival in human mammary carcinoma patients. A random forest analysis suggested predictive value of the complete set of differentially expressed genes between early- and late-stage CAFs on bulk tumor patient samples, supporting the clinical relevance of our findings. In conclusion, our data show transcriptome alterations in CAFs during tumorigenesis in the mammary gland, which suggest that CAFs are educated by the tumor over time to promote tumor development. Moreover, we show that murine CAF gene signatures can harbor predictive value for human cancer.
uORF-tools—workflow for the determination of translation-regulatory upstream open reading frames
(2019)
Ribosome profiling (ribo-seq) provides a means to analyze active translation by determining ribosome occupancy in a transcriptome-wide manner. The vast majority of ribosome protected fragments (RPFs) resides within the protein-coding sequence of mRNAs. However, commonly reads are also found within the transcript leader sequence (TLS) (aka 5’ untranslated region) preceding the main open reading frame (ORF), indicating the translation of regulatory upstream ORFs (uORFs). Here, we present a workflow for the identification of translation-regulatory uORFs. Specifically, uORF-Tools uses Ribo-TISH to identify uORFs within a given dataset and generates a uORF annotation file. In addition, a comprehensive human uORF annotation file, based on 35 ribo-seq files, is provided, which can serve as an alternative input file for the workflow. To assess the translation-regulatory activity of the uORFs, stimulus-induced changes in the ratio of the RPFs residing in the main ORFs relative to those found in the associated uORFs are determined. The resulting output file allows for the easy identification of candidate uORFs, which have translation-inhibitory effects on their associated main ORFs. uORF-Tools is available as a free and open Snakemake workflow at https://github.com/Biochemistry1-FFM/uORF-Tools. It is easily installed and all necessary tools are provided in a version-controlled manner, which also ensures lasting usability. uORF-Tools is designed for intuitive use and requires only limited computing times and resources.
Despite the success of immune checkpoint blockade in cancer, the number of patients that benefit from this revolutionary treatment option remains low. Therefore, efforts are being undertaken to sensitize tumors for immune checkpoint blockade, which includes combining immune checkpoint blocking agents such as anti-PD-1 antibodies with standard of care treatments. Here we report that a combination of chemotherapy (doxorubicin) and immune checkpoint blockade (anti-PD-1 antibodies) induces superior tumor control compared to chemotherapy and immune checkpoint blockade alone in the murine autochthonous polyoma middle T oncogene-driven (PyMT) mammary tumor model. Using whole transcriptome analysis, we identified a set of genes that were upregulated specifically upon chemoimmunotherapy. This gene signature and, more specifically, a condensed four-gene signature predicted favorable survival of human mammary carcinoma patients in the METABRIC cohort. Moreover, PyMT tumors treated with chemoimmunotherapy contained higher levels of cytotoxic lymphocytes, particularly natural killer cells (NK cells). Gene set enrichment analysis and bead-based ELISA measurements revealed increased IL-27 production and signaling in PyMT tumors upon chemoimmunotherapy. Moreover, IL-27 signaling improved NK cell cytotoxicity against PyMT cells in vitro. Taken together, our data support recent clinical observations indicating a benefit of chemoimmunotherapy compared to monotherapy in breast cancer and suggest potential underlying mechanisms.
Macrophages constitute a major part of the tumor-infiltrating immune cells. Within the tumor microenvironment, they acquire an alternatively activated, tumor-supporting phenotype. Factors released by tumor cells are crucial for the recruitment of tumor-associated macrophages. In the present project, we aimed to understand the role of hsa-miR-200c-3p (miR-200c) in the interplay between tumor cells and macrophages. To this end, we employed a coculture system of MCF7 breast tumor cells and primary human macrophages and observed the transfer of miR-200c from apoptotic tumor cells to macrophages, which required intact CD36 receptor in macrophages. We further comprehensively determined miR-200c targets in macrophages by mRNA-sequencing and identified numerous migration-associated mRNAs to be downregulated by miR-200c. Consequently, miR-200c attenuated macrophage infiltration into 3-dimensional tumor spheroids. miR-200c-mediated reduction in infiltration further correlated with a miR-200c migration signature comprised of the four miR-200c-repressed, predicted targets PPM1F, RAB11FIB2, RDX, and MSN.
Macrophages acquire anti-inflammatory and proresolving functions to facilitate resolution of inflammation and promote tissue repair. While alternatively activated macrophages (AAMs), also referred to as M2 macrophages, polarized by type 2 (Th2) cytokines IL-4 or IL-13 contribute to the suppression of inflammatory responses and play a pivotal role in wound healing, contemporaneous exposure to apoptotic cells (ACs) potentiates the expression of anti-inflammatory and tissue repair genes. Given that liver X receptors (LXRs), which coordinate sterol metabolism and immune cell function, play an essential role in the clearance of ACs, we investigated whether LXR activation following engulfment of ACs selectively potentiates the expression of Th2 cytokine-dependent genes in primary human AAMs. We show that AC uptake simultaneously upregulates LXR-dependent, but suppresses SREBP-2-dependent gene expression in macrophages, which are both prevented by inhibiting Niemann–Pick C1 (NPC1)-mediated sterol transport from lysosomes. Concurrently, macrophages accumulate sterol biosynthetic intermediates desmosterol, lathosterol, lanosterol, and dihydrolanosterol but not cholesterol-derived oxysterols. Using global transcriptome analysis, we identify anti-inflammatory and proresolving genes including interleukin-1 receptor antagonist (IL1RN) and arachidonate 15-lipoxygenase (ALOX15) whose expression are selectively potentiated in macrophages upon concomitant exposure to ACs or LXR agonist T0901317 (T09) and Th2 cytokines. We show priming macrophages via LXR activation enhances the cellular capacity to synthesize inflammation-suppressing specialized proresolving mediator (SPM) precursors 15-HETE and 17-HDHA as well as resolvin D5. Silencing LXRα and LXRβ in macrophages attenuates the potentiation of ALOX15 expression by concomitant stimulation of ACs or T09 and IL-13. Collectively, we identify a previously unrecognized mechanism of regulation whereby LXR integrates AC uptake to selectively shape Th2-dependent gene expression in AAMs.
Tolerizing CTL by sustained hepatic PD-L1 expression provides a new therapy spproach in mouse sepsis
(2019)
Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option.
Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate- and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection.
Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers.
Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic, Th17-derived cytokine thought to critically contribute to the pathogenesis of diverse autoimmune diseases, including rheumatoid arthritis and psoriasis. Treatment with monoclonal antibodies that block GM-CSF activity is associated with favorable therapeutic effects in patients with rheumatoid arthritis. We evaluated the role of GM-CSF as a potential target for therapeutic interference in psoriasis using a combined pharmacologic and genetic approach and the mouse model of imiquimod-induced psoriasiform dermatitis (IMQPD). Neutralization of murine GM-CSF by an anti-GM-CSF antibody ameliorated IMQPD. In contrast, genetic deficiency in GM-CSF did not alter the course of IMQPD, suggesting the existence of mechanisms compensating for chronic, but not acute, absence of GM-CSF. Further investigation uncovered an alternative pathogenic pathway for IMQPD in the absence of GM-CSF characterized by an expanded plasmacytoid dendritic cell population and release of IFNα and IL-22. This pathway was not activated in wild-type mice during short-term anti-GM-CSF treatment. Our investigations support the potential value of GM-CSF as a therapeutic target in psoriatic disease. The discovery of an alternative pathogenic pathway for psoriasiform dermatitis in the permanent absence of GM-CSF, however, suggests the need for monitoring during therapeutic use of long-term GM-CSF blockade.
5-Lipoxygenase contributes to PPAR [gamma] activation in macrophages in response to apoptotic cells
(2012)
Background: One hallmark contributing to immune suppression during the late phase of sepsis is macrophage polarization to an anti-inflammatory phenotype upon contact with apoptotic cells (AC). Taking the important role of the nuclear receptor PPARγ for this phenotype switch into consideration, it remains elusive how AC activate PPARγ in macrophages. Therefore, we were interested to characterize the underlying principle.
Methods: Apoptosis was induced by treatment of Jurkat T cells for 3 hours with 0.5 μg/ml staurosporine. Necrotic cells (NC) were prepared by heating cells for 20 minutes to 65°C. PPARγ activation was followed by stably transducing RAW264.7 macrophages with a vector encoding the red fluorescent protein mRuby after PPARγ binding to 4 × PPRE sites downstream of the reporter gene sequence. This readout was established by treatment with the PPARγ agonist rosiglitazone (1 μM) and AC (5:1). Twenty-four hours after stimulation, mRuby expression was analysed by fluorescence microscopy. Lipid rafts of AC, NC, as well as living cells (LC) were enriched by sucrose gradient centrifugation. Fractions were analysed for lipid raft-associated marker proteins. Lipid rafts were incubated with transduced RAW264.7 macrophages as described above. 5-Lipoxygenase (5-LO) involvement was verified by pharmacological inhibition (MK-866, 1 μM) and overexpression.
Results: Assuming that the molecule responsible for PPARγ activation in macrophages is localized in the cell membrane of AC, most probably associated to lipid rafts, we isolated lipid rafts from AC, NC and LC. Mass spectrometric analysis of lipid rafts of AC showed the expression of 5-LO, whereas lipid rafts of LC did not. Moreover, incubating macrophages with lipid rafts of AC induced mRuby expression. In contrast, lipid rafts of NC and LC did not. To verify the involvement of 5-LO in activating PPARγ in macrophages, Jurkat T cells were incubated for 30 minutes with the 5-LO inhibitor MK-866 (1 μM) before apoptosis induction. In line with our hypothesis, these AC did not induce mRuby expression. Finally, although living Jurkat T cells overexpressing 5-LO did not activate PPARγ in macrophages, mRuby expression was significantly increased when AC were generated from 5-LO overexpressing compared with wild-type Jurkat cells.
Conclusion: Our results suggest that induction of apoptosis activates 5-LO, localizing to lipid rafts, necessary for PPARγ activation in macrophages. Therefore, it will be challenging to determine whether 5-LO activity in AC, generated from other cell types, correlates with PPARγ activation, contributing to an immune-suppressed phenotype in macrophages.
We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.
Peroxisome proliferator-activated receptor γ (PPARγ) gained considerable interest as a therapeutic target during chronic inflammatory diseases. Remarkably, the pathogenesis of diseases such as multiple sclerosis or Alzheimer is associated with impaired PPARγ expression. Considering that regulation of PPARγ expression during inflammation is largely unknown, we were interested in elucidating underlying mechanisms. To this end, we initiated an inflammatory response by exposing primary human macrophages to lipopolysaccharide (LPS) and observed a rapid decline of PPARγ1 expression. Because promoter activities were not affected by LPS, we focused on mRNA stability and noticed a decreased mRNA half-life. As RNA stability is often regulated via 3′-untranslated regions (UTRs), we analyzed the impact of the PPARγ-3′-UTR by reporter assays using specific constructs. LPS significantly reduced luciferase activity of the pGL3-PPARγ-3′-UTR, suggesting that PPARγ1 mRNA is destabilized. Deletion or mutation of a potential microRNA-27a/b (miR-27a/b) binding site within the 3′-UTR restored luciferase activity. Moreover, inhibition of miR-27b, which was induced upon LPS exposure, partially reversed PPARγ1 mRNA decay, whereas miR-27b overexpression decreased PPARγ1 mRNA content. In addition, LPS further reduced this decay. The functional relevance of miR-27b-dependent PPARγ1 decrease was proven by inhibition or overexpression of miR-27b, which affected LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin (IL)-6. We provide evidence that LPS-induced miR-27b contributes to destabilization of PPARγ1 mRNA. Understanding molecular mechanisms decreasing PPARγ might help to better appreciate inflammatory diseases.
Macrophages ingesting apoptotic cells attenuate inflammatory responses, such as reactive oxygen species (ROS) generation. In atherosclerosis, ongoing inflammation and accumulation of apoptotic/necrotic material are observed, suggesting defects of phagocytes in recognizing or responding to dying cells. Modified lipoproteins such as oxidized LDL (oxLDL) are known to promote inflammation and to interfere with apoptotic cell clearance. Here, we studied the impact of cells exposed to oxLDL on their ability to interfere with the oxidative burst in phagocytes. In contrast to apoptotic cells, cells dying in response to or in the presence of oxLDL failed to suppress ROS generation despite efficiently being taken up by phagocytes. In addition, apoptotic cells, but not oxLDL-treated cells, inhibited phosphorylation of extracellular signal-regulated kinase, which is important for NADPH oxidase activation. oxLDL treatment did not interfere with activation of the antiinflammatory transcriptional regulator peroxisome proliferator-activated receptor gamma by apoptotic cells. Moreover, cells exposed to oxLDL failed to suppress lipopolysaccharide- induced proinflammatory cytokine expression, whereas apoptotic cells attenuated these phagocyte responses. Thus, the presence of oxLDL during cell death impaired the ability of apoptotic cells to act antiinflammatory with regard to oxidative burst inhibition and cytokine expression in phagocytes.
Prostaglandin E2 (PGE2) favors multiple aspects of tumor development and immune evasion. Therefore, microsomal prostaglandin E synthase (mPGES-1/-2), is a potential target for cancer therapy. We explored whether inhibiting mPGES-1 in human and mouse models of breast cancer affects tumor-associated immunity. A new model of breast tumor spheroid killing by human PBMCs was developed. In this model, tumor killing required CD80 expression by tumor-associated phagocytes to trigger cytotoxic T cell activation. Pharmacological mPGES-1 inhibition increased CD80 expression, whereas addition of PGE2, a prostaglandin E2 receptor 2 (EP2) agonist, or activation of signaling downstream of EP2 reduced CD80 expression. Genetic ablation of mPGES-1 resulted in markedly reduced tumor growth in PyMT mice. Macrophages of mPGES-1-/- PyMT mice indeed expressed elevated levels of CD80 compared to their wildtype counterparts. CD80 expression in tumor-spheroid infiltrating mPGES-1-/- macrophages translated into antigen-specific cytotoxic T cell activation. In conclusion, mPGES-1 inhibition elevates CD80 expression by tumor-associated phagocytes to restrict tumor growth. We propose that mPGES-1 inhibition in combination with immune cell activation might be part of a therapeutic strategy to overcome the immunosuppressive tumor microenvironment.
This study indicates that embryonic stem cells [ESCs] cultured with retinoic acid and activin A significantly upregulate the miRNA let-7e. This specific miRNA modulates the Wnt pathway and the expression of early nephrogenic markers under these differentiation conditions. The differentiation markers WT1, Pax2 and Wnt4 were downregulated when miRNA let-7e was silenced, thus indicating the role of miRNA let-7e in the differentiation process. PKCβ, GSK3β phosphorylation (GSK3βP) and β-catenin expression was reduced in differentiated cells and reversed by miRNA let-7e silencing. Addition of a PKCβ inhibitor to the miRNA let-7e silenced cells abolished let-7e-derived effects in differentiation markers, and reversed the increase in GSK3βP and β-catenin, thus indicating that miRNA let-7e is involved in differentiation via the modulation of GSK3β phosphorylation and β-catenin production.
miRNA let-7e is involved in stem cell differentiation, and metalloproteinases are among its potential target genes. We hypothesized that the inhibitory action of let-7e on regulation of MMP9 expression could represent a crucial mechanism during differentiation of adipose-derived stem cells (ASCs). ASCs were differentiated with all-trans retinoic acid (ATRA) to promote differentiation, and the effect of let-7 silencing during differentiation was tested. Results indicate that ASCs cultured with ATRA differentiated into cells of the epithelial lineage. We found that ASCs cultured with ATRA or transfected with miRNA let-7e expressed epithelial markers such as cytokeratin-18 and early renal organogenesis markers such as Pax2, Wt1, Wnt4 and megalin. Conversely, the specific knockdown of miRNA let-7e in ASCs significantly decreased the expression of these genes, indicating its vital role during the differentiation process. Using luciferase reporter assays, we also showed that MMP9 is a direct target of miRNA let-7e. Thus, our results suggest that miRNA let-7e acts as a matrix metalloproteinase-9 (MMP9) inhibitor and differentiation inducer in ASCs.
Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.
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.
Aims: Parkinson's disease (PD) is frequently associated with a prodromal sensory neuropathy manifesting with sensory loss and chronic pain. We have recently shown that PD-associated sensory neuropathy in patients is associated with high levels of glucosylceramides. Here, we assessed the underlying pathology and mechanisms in Pink1−/−SNCAA53T double mutant mice. Methods: We studied nociceptive and olfactory behaviour and the neuropathology of dorsal root ganglia (DRGs), including ultrastructure, mitochondrial respiration, transcriptomes, outgrowth and calcium currents of primary neurons, and tissue ceramides and sphingolipids before the onset of a PD-like disease that spontaneously develops in Pink1−/−SNCAA53T double mutant mice beyond 15 months of age. Results: Similar to PD patients, Pink1−/−SNCAA53T mice developed a progressive prodromal sensory neuropathy with a loss of thermal sensitivity starting as early as 4 months of age. In analogy to human plasma, lipid analyses revealed an accumulation of glucosylceramides (GlcCer) in the DRGs and sciatic nerves, which was associated with pathological mitochondria, impairment of mitochondrial respiration, and deregulation of transient receptor potential channels (TRPV and TRPA) at mRNA, protein and functional levels in DRGs. Direct exposure of DRG neurons to GlcCer caused transient hyperexcitability, followed by a premature decline of the viability of sensory neurons cultures upon repeated GlcCer application. Conclusions: The results suggest that pathological GlcCer contribute to prodromal sensory disease in PD mice via mitochondrial damage and calcium channel hyperexcitability. GlcCer-associated sensory neuron pathology might be amenable to GlcCer lowering therapeutic strategies.
The interaction of macrophages with apoptotic cells is required for efficient resolution of inflammation. While apoptotic cell removal prevents inflammation due to secondary necrosis, it also alters the macrophage phenotype to hinder further inflammatory reactions. The interaction between apoptotic cells and macrophages is often studied by chemical or biological induction of apoptosis, which may introduce artifacts by affecting the macrophages as well and/or triggering unrelated signaling pathways. Here, we set up a pure cell death system in which NIH 3T3 cells expressing dimerizable Caspase-8 were co-cultured with peritoneal macrophages in a transwell system. Phenotype changes in macrophages induced by apoptotic cells were evaluated by RNA sequencing, which revealed an unexpectedly dominant impact on macrophage proliferation. This was confirmed in functional assays with primary peritoneal macrophages and IC-21 macrophages. Moreover, inhibition of apoptosis during Zymosan-induced peritonitis in mice decreased mRNA levels of cell cycle mediators in peritoneal macrophages. Proliferation of macrophages in response to apoptotic cells may be important to increase macrophage numbers in order to allow efficient clearance and resolution of inflammation.
IL-27 regulates inflammatory diseases by exerting a pleiotropic impact on immune cells. In cancer, IL-27 restricts tumor growth by acting on tumor cells directly, while its role in the tumor microenvironment is still controversially discussed. To explore IL-27 signaling in the tumor stroma, we used a mammary carcinoma syngraft approach in IL27Rα-deficient mice. Tumor growth in animals lacking IL27Rα was markedly reduced. We noticed a decrease in immune cell infiltrates, enhanced tumor cell death, and fibroblast accumulation. However, most striking changes pertain the tumor vasculature. Tumors in IL27Rα-deficient mice were unable to form functional vessels. Blocking IL-27-STAT1 signaling in endothelial cells in vitro provoked an overshooting migration/sprouting of endothelial cells. Apparently, the lack of the IL-27 receptor caused endothelial cell hyper-activation via STAT1 that limited vessel maturation. Our data reveal a so far unappreciated role of IL-27 in endothelial cells with importance in pathological vessel formation.
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.
Macrophages respond to the Th2 cytokine IL-4 with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibited IL-4-evoked activation of STAT3 while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. In addition, phenformin prevented IL-4-induced association of STAT6 and Lys-9 acetylation of histone H3 at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by ALOX15 knockdown. Finally, pretreatment of macrophages with IL-4 for 48 h increased the mRNA expression of the proinflammatory cytokines IL-6, IL-12, CXCL9, and CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubation with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages.
Hypoxia potentiates palmitate-induced pro-inflammatory activation of primary human macrophages
(2015)
Pro-inflammatory cytokines secreted by adipose tissue macrophages (ATMs) contribute to chronic low-grade inflammation and obesity-induced insulin resistance. Recent studies have shown that adipose tissue hypoxia promotes an inflammatory phenotype in ATMs. However, our understanding of how hypoxia modulates the response of ATMs to free fatty acids within obese adipose tissue is limited. We examined the effects of hypoxia (1% O2) on the pro-inflammatory responses of human monocyte-derived macrophages to the saturated fatty acid palmitate. Compared with normoxia, hypoxia significantly increased palmitate-induced mRNA expression and protein secretion of IL-6 and IL-1β. Although palmitate-induced endoplasmic reticulum stress and nuclear factor κB pathway activation were not enhanced by hypoxia, hypoxia increased the activation of JNK and p38 mitogen-activated protein kinase signaling in palmitate-treated cells. Inhibition of JNK blocked the hypoxic induction of pro-inflammatory cytokine expression, whereas knockdown of hypoxia-induced transcription factors HIF-1α and HIF-2α alone or in combination failed to reduce IL-6 and only modestly reduced IL-1β gene expression in palmitate-treated hypoxic macrophages. Enhanced pro-inflammatory cytokine production and JNK activity under hypoxia were prevented by inhibiting reactive oxygen species generation. In addition, silencing of dual-specificity phosphatase 16 increased normoxic levels of IL-6 and IL-1β and reduced the hypoxic potentiation in palmitate-treated macrophages. The secretome of hypoxic palmitate-treated macrophages promoted IL-6 and macrophage chemoattractant protein 1 expression in primary human adipocytes, which was sensitive to macrophage JNK inhibition. Our results reveal that the coexistence of hypoxia along with free fatty acids exacerbates macrophage-mediated inflammation.
Background: Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells.
Aims: We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets.
Methods: Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used.
Results: Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented.
Conclusions: We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth.
Hypoxia inhibits ferritinophagy, increases mitochondrial ferritin, and protects from ferroptosis
(2020)
Highlights
• Hypoxia decreases NCOA4 transcription in primary human macrophages.
• NCOA4 mRNA is a target of miR-6862-5p.
• Lowering NCOA4 increases FTMT abundance under hypoxia.
• FTMT and FTH protect from ferroptosis.
• Tumor cells lack the hypoxic decrease of NCOA4 and fail to stabilize FTMT.
Abstract
Cellular iron, at the physiological level, is essential to maintain several metabolic pathways, while an excess of free iron may cause oxidative damage and/or provoke cell death. Consequently, iron homeostasis has to be tightly controlled. Under hypoxia these regulatory mechanisms for human macrophages are not well understood. Hypoxic primary human macrophages reduced intracellular free iron and increased ferritin expression, including mitochondrial ferritin (FTMT), to store iron. In parallel, nuclear receptor coactivator 4 (NCOA4), a master regulator of ferritinophagy, decreased and was proven to directly regulate FTMT expression. Reduced NCOA4 expression resulted from a lower rate of hypoxic NCOA4 transcription combined with a micro RNA 6862-5p-dependent degradation of NCOA4 mRNA, the latter being regulated by c-jun N-terminal kinase (JNK). Pharmacological inhibition of JNK under hypoxia increased NCOA4 and prevented FTMT induction. FTMT and ferritin heavy chain (FTH) cooperated to protect macrophages from RSL-3-induced ferroptosis under hypoxia as this form of cell death is linked to iron metabolism. In contrast, in HT1080 fibrosarcome cells, which are sensitive to ferroptosis, NCOA4 and FTMT are not regulated. Our study helps to understand mechanisms of hypoxic FTMT regulation and to link ferritinophagy and macrophage sensitivity to ferroptosis.
Background: Glucose metabolism in the tumor-microenvironment is a fundamental hallmark for tumor growth and intervention therein remains an attractive option for anti-tumor therapy. Whether tumor-derived factors such as microRNAs (miRs) regulate glucose metabolism in stromal cells, especially in tumor-associated macrophages (TAMs), to hijack them for trophic support, remains elusive.
Methods: Ago-RIP-Seq identified macrophage lactate dehydrogenase B (LDHB) as a target of tumor-derived miR-375 in both 2D/3D cocultures and in murine TAMs from a xenograft mouse model. The prognostic value was analyzed by ISH and multiplex IHC of breast cancer patient tissues. Functional consequences of the miR-375-LDHB axis in TAMs were investigated upon mimic/antagomir treatment by live metabolic flux assays, GC/MS, qPCR, Western blot, lentiviral knockdown and FACS. The therapeutic potential of a combinatorial miR-375-decoy/simvastatin treatment was validated by live cell imaging.
Results: Macrophage LDHB decreased in murine and human breast carcinoma. LDHB downregulation increase aerobic glycolysis and lactagenesis in TAMs in response to tumor-derived miR-375. Lactagenesis reduced fatty acid synthesis but activated SREBP2, which enhanced cholesterol biosynthesis in macrophages. LDHB downregulation skewed TAMs to function as a lactate and sterol/oxysterol source for the proliferation of tumor cells. Restoring of LDHB expression potentiated inhibitory effects of simvastatin on tumor cell proliferation.
Conclusion: Our findings identified a crucial role of LDHB in macrophages and established tumor-derived miR-375 as a novel regulator of macrophage metabolism in breast cancer, which might pave the way for strategies of combinatorial cancer cell/stroma cell interventions.
The lung tumor microenvironment plays a critical role in the tumorigenesis and metastasis of lung cancer, resulting from the crosstalk between cancer cells and microenvironmental cells. Therefore, comprehensive identification and characterization of cell populations in the complex lung structure is crucial for development of novel targeted anti-cancer therapies. Here, a hierarchical clustering approach with multispectral flow cytometry was established to delineate the cellular landscape of murine lungs under steady-state and cancer conditions. Fluorochromes were used multiple times to be able to measure 24 cell surface markers with only 13 detectors, yielding a broad picture for whole-lung phenotyping. Primary and metastatic murine lung tumor models were included to detect major cell populations in the lung, and to identify alterations to the distribution patterns in these models. In the primary tumor models, major altered populations included CD324+ epithelial cells, alveolar macrophages, dendritic cells, and blood and lymph endothelial cells. The number of fibroblasts, vascular smooth muscle cells, monocytes (Ly6C+ and Ly6C–) and neutrophils were elevated in metastatic models of lung cancer. Thus, the proposed clustering approach is a promising method to resolve cell populations from complex organs in detail even with basic flow cytometers.
Bacterial and fungal toll-like receptor activation elicits type I IFN responses in mast cells
(2021)
Next to their role in IgE-mediated allergic diseases and in promoting inflammation, mast cells also have antiinflammatory functions. They release pro- as well as antiinflammatory mediators, depending on the biological setting. Here we aimed to better understand the role of mast cells during the resolution phase of a local inflammation induced with the Toll-like receptor (TLR)-2 agonist zymosan. Multiple sequential immunohistology combined with a statistical neighborhood analysis showed that mast cells are located in a predominantly antiinflammatory microenvironment during resolution of inflammation and that mast cell-deficiency causes decreased efferocytosis in the resolution phase. Accordingly, FACS analysis showed decreased phagocytosis of zymosan and neutrophils by macrophages in mast cell-deficient mice. mRNA sequencing using zymosan-induced bone marrow-derived mast cells (BMMC) revealed a strong type I interferon (IFN) response, which is known to enhance phagocytosis by macrophages. Both, zymosan and lipopolysaccharides (LPS) induced IFN-β synthesis in BMMCs in similar amounts as in bone marrow derived macrophages. IFN-β was expressed by mast cells in paws from naïve mice and during zymosan-induced inflammation. As described for macrophages the release of type I IFNs from mast cells depended on TLR internalization and endosome acidification. In conclusion, mast cells are able to produce several mediators including IFN-β, which are alone or in combination with each other able to regulate the phagocytotic activity of macrophages during resolution of inflammation.