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BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer, and despite low incidence rates, it remains the sixth leading cause of cancer related deaths worldwide. Immunotherapy, which aims to enhance the immune system’s ability to recognize and eliminate cancer cells, has emerged as a promising approach in the battle against PDAC. PARP7, a mono-ADP-ribosyltransferase, is a negative regulator of the type I interferon (IFN-I) pathway and has been reported to reduce anti-tumour immunity.MethodsWe used murine pancreatic cancer cells, CR705, CRISPR/Cas9, in vivo tumour models and spectral flow cytometry to determine the role of PARP7 in pancreatic tumour growth.ResultsLoss of Parp7 elevated the levels of interferon stimulated gene factor 3 (ISGF3) and its downstream target genes, even in the absence of STING. Cancer cells knocked out for Parp7 (CR705Parp7KO) produced smaller tumours than control cells (CR705Cas9) when injected into immunocompetent mice. Transcriptomic analyses revealed that CR705Parp7KO tumours had increased expression of genes involved in immunoregulatory interactions and interferon signalling pathways. Characterization of tumour infiltrating leukocyte (TIL) populations showed that CR705Parp7KO tumours had higher proportions of natural killer cells, CD8+ T cells and a lower proportion of anti-inflammatory macrophages (M2). The overall TIL profile of CR705Parp7KO tumours was suggestive of a less suppressive microenvironment.ConclusionsOur data show that loss of Parp7 reduces PDAC tumour growth by increasing the infiltration of immune cells and enhancing anti-tumour immunity. These findings provide support to pursue PARP7 as a therapeutic target for cancer treatment.

The aryl hydrocarbon receptor (AHR) is a ligand activated transcription factor which in certain cancer types drives pro-survival processes that facilitate tumorigenesis, malignant cell migration, invasion, and metastasis. Much of AHR’s pro-tumorigenic action is due to its activation by the oncometabolite, kynurenine. Because of this AHR antagonists are being actively investigated as new anti-tumor therapy. In this study we compared the effects of treatment with the AHR antagonists, BAY2416964 and GNF351, to that of AHR knockout in PyMT murine mammary cancer cells. BAY2416964 and GNF351 effectively inhibited kynurenine-dependent increases in Cyp1a1 and Cyp1b1 mRNA levels. CRISPR/Cas9-generated PyMT AhrKO cells exhibited reduced cell proliferation compared with controls, but treatment with 1 μM BAY2416964 for 96 h had no effect on the proliferation of wildtype cells. To further examine the differences between AHR knockout and short term BAY2416964, we generated long-term BAY2416964 (LT-BAY) cells by exposing wildtype cells to 1 μM BAY2416964 for at least 6 weeks. Similar to AhrKO cells, LT-BAY cells exhibited reduced cell proliferation and migration compared with wildtype cells. No differentially expressed genes (DEGs) were identified in wildtype cells exposed to 1 μM BAY2416964 for 24 h; however, 46.4% of DEGs overlapped between AhrKO and LT-BAY cells including gene regulated cell proliferation. Our data reveal long-term pharmacological inhibition of AHR by BAY2416964 closely resembles AHR loss in a mouse model of breast cancer.

Abstract Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract. A clear gap in our existing CD diagnostics and current disease management approaches is the lack of highly specific biomarkers that can be used to streamline or personalize disease management. Comprehensive profiling of metabolites holds promise; however, these high-dimensional profiles need to be reduced to have relevance in the context of CD. Machine learning approaches are optimally suited to bridge this gap in knowledge by contextualizing the metabolic alterations in CD using genome-scale metabolic network reconstructions. Our work presents a framework for studying altered metabolic reactions between patients with CD and controls using publicly available transcriptomic data and existing gene-driven metabolic network reconstructions. Additionally, we apply the same methods to patient-derived ileal enteroids to explore the utility of using this experimental in vitro platform for studying CD. Furthermore, we have piloted an untargeted metabolomics approach as a proof-of-concept validation strategy in human ileal mucosal tissue. These findings suggest that in silico metabolic modeling can potentially identify pathways of clinical relevance in CD, paving the way for the future discovery of novel diagnostic biomarkers and therapeutic targets.

Background Colorectal cancer (CRC) has a high mortality rate and can develop in either colitis-dependent (colitis-associated (CA)-CRC) or colitis-independent (sporadic (s)CRC) manner. There has been a significant debate about whether mast cells (MCs) promote or inhibit the development of CRC. Herein we investigated MC activity throughout the multistepped development of CRC in both human patients and animal models.Methods We analyzed human patient matched samples of healthy colon vs CRC tissue alongside conducting a The Cancer Genome Atlas-based immunogenomic analysis and multiple experiments employing genetically engineered mouse (GEM) models.Results Analyzing human CRC samples revealed that MCs can be active or inactive in this disease. An activated MC population decreased the number of tumor-residing CD8 T cells. In mice, MC deficiency decreased the development of CA-CRC lesions, while it increased the density of tumor-based CD8 infiltration. Furthermore, co-culture experiments revealed that tumor-primed MCs promote apoptosis in CRC cells. In MC-deficient mice, we found that MCs inhibited the development of sCRC lesions. Further exploration of this with several GEM models confirmed that different immune responses alter and are altered by MC activity, which directly alters colon tumorigenesis. Since rescuing MC activity with bone marrow transplantation in MC-deficient mice or pharmacologically inhibiting MC effects impacts the development of sCRC lesions, we explored its therapeutic potential against CRC. MC activity promoted CRC cell engraftment by inhibiting CD8+ cell infiltration in tumors, pharmacologically blocking it inhibits the ability of allograft tumors to develop. This therapeutic strategy potentiated the cytotoxic activity of fluorouracil chemotherapy.Conclusion Therefore, we suggest that MCs have a dual role throughout CRC development and are potential druggable targets against this disease.

Abstract Background Hypoxia is pervasive in cancer and other diseases. Cells sense and adapt to hypoxia by activating hypoxia-inducible transcription factors (HIFs), but it is still an outstanding question why cell types differ in their transcriptional response to hypoxia. Results We report that HIFs fail to bind CpG dinucleotides that are methylated in their consensus binding sequence, both in in vitro biochemical binding assays and in vivo studies of differentially methylated isogenic cell lines. Based on in silico structural modeling, we show that 5-methylcytosine indeed causes steric hindrance in the HIF binding pocket. A model wherein cell-type-specific methylation landscapes, as laid down by the differential expression and binding of other transcription factors under normoxia, control cell-type-specific hypoxia responses is observed. We also discover ectopic HIF binding sites in repeat regions which are normally methylated. Genetic and pharmacological DNA demethylation, but also cancer-associated DNA hypomethylation, expose these binding sites, inducing HIF-dependent expression of cryptic transcripts. In line with such cryptic transcripts being more prone to cause double-stranded RNA and viral mimicry, we observe low DNA methylation and high cryptic transcript expression in tumors with high immune checkpoint expression, but not in tumors with low immune checkpoint expression, where they would compromise tumor immunotolerance. In a low-immunogenic tumor model, DNA demethylation upregulates cryptic transcript expression in a HIF-dependent manner, causing immune activation and reducing tumor growth. Conclusions Our data elucidate the mechanism underlying cell-type-specific responses to hypoxia and suggest DNA methylation and hypoxia to underlie tumor immunotolerance.

Poly(ADP-ribose) polymerase 7 (PARP-7) has emerged as a critically important member of a large enzyme family that catalyzes ADP-ribosylation in mammalian cells. PARP-7 is a critical regulator of the innate immune response. What remains unclear is the mechanism by which PARP-7 regulates this process, namely because the protein targets of PARP-7 mono-ADP-ribosylation (MARylation) are largely unknown. Here, we combine chemical genetics, proximity labeling, and proteome-wide amino acid ADP-ribosylation site profiling for identifying the direct targets and sites of PARP-7-mediated MARylation in a cellular context. We found that the inactive PARP family member, PARP-13—a critical regulator of the antiviral innate immune response—is a major target of PARP-7. PARP-13 is preferentially MARylated on cysteine residues in its RNA binding zinc finger domain. Proteome-wide ADP-ribosylation analysis reveals cysteine as a major MARylation acceptor of PARP-7. This study provides insight into PARP-7 targeting and MARylation site preference.

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Apart from its role in the metabolism of carcinogens, the aryl hydrocarbon receptor (AhR) has been suggested to be involved in the control of inflammatory responses within the respiratory tract. However, the mechanisms responsible for this are only partially known. In this study, we used A549 cell line, as a human model of lung alveolar type II (ATII)-like cells, to study the functional role of the AhR in control of inflammatory responses. Using IL-1β as an inflammation inducer, we found that the induction of cyclooxygenase-2 and secretion of prostaglandins, as well as expression and release of pro-inflammatory cytokines, were significantly higher in the AhR-deficient A549 cells. This was linked with an increased nuclear factor-κB (NF-κB) activity, and significantly enhanced phosphorylation of its regulators, IKKα/β, and their target IκBα, in the AhR-deficient A549 cells. In line with this, when we mimicked the exposure to a complex mixture of airborne pollutants, using an organic extract of reference diesel exhaust particle mixture, an exacerbated inflammatory response was observed in the AhR-deficient cells, as compared with wild-type A549 cells. Together, the present results indicate that the AhR may act as a negative regulator of the inflammatory response in the A549 model, via a direct modulation of NF-κB signaling. Its role(s) in the control of inflammation within the lung alveoli exposed to airborne pollutants, especially those which simultaneously activate the AhR, thus deserve further attention.

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ADP-ribosylation is a post-translational protein modification catalyzed by a family of proteins known as poly-ADP-ribose polymerases. PARP7 (TIPARP; ARTD14) is a mono-ADP-ribosyltransferase involved in several cellular processes, including responses to hypoxia, innate immunity and regulation of nuclear receptors. Since previous studies suggested that PARP7 was regulated by 17β-estradiol, we investigated whether PARP7 regulates estrogen receptor α signaling. We confirmed the 17β-estradiol-dependent increases of PARP7 mRNA and protein levels in MCF-7 cells, and observed recruitment of estrogen receptor α to the promoter of PARP7. Overexpression of PARP7 decreased ligand-dependent estrogen receptor α signaling, while treatment of PARP7 knockout MCF-7 cells with 17β-estradiol resulted in increased expression of and recruitment to estrogen receptor α target genes, in addition to increased proliferation. Co-immunoprecipitation assays revealed that PARP7 mono-ADP-ribosylated estrogen receptor α, and mass spectrometry mapped the modified peptides to the receptor’s ligand-independent transactivation domain. Co-immunoprecipitation with truncated estrogen receptor α variants identified that the hinge region of the receptor is required for PARP7-dependent mono-ADP-ribosylation. These results imply that PARP7-mediated mono-ADP-ribosylation may play an important role in estrogen receptor positive breast cancer.

The cholesterol-sensing nuclear receptor liver X receptor (LXR) and the glucose-sensing transcription factor carbohydrate responsive element-binding protein (ChREBP) are central players in regulating glucose and lipid metabolism in the liver. More knowledge of their mechanistic interplay is needed to understand their role in pathological conditions like fatty liver disease and insulin resistance. In the current study, LXR and ChREBP co-occupancy was examined by analyzing ChIP-seq datasets from mice livers. LXR and ChREBP interaction was determined by Co-immunoprecipitation (CoIP) and their transactivity was assessed by real-time quantitative polymerase chain reaction (qPCR) of target genes and gene reporter assays. Chromatin binding capacity was determined by ChIP-qPCR assays. Our data show that LXRα and ChREBPα interact physically and show a high co-occupancy at regulatory regions in the mouse genome. LXRα co-activates ChREBPα and regulates ChREBP-specific target genes in vitro and in vivo. This co-activation is dependent on functional recognition elements for ChREBP but not for LXR, indicating that ChREBPα recruits LXRα to chromatin in trans. The two factors interact via their key activation domains; the low glucose inhibitory domain (LID) of ChREBPα and the ligand-binding domain (LBD) of LXRα. While unliganded LXRα co-activates ChREBPα, ligand-bound LXRα surprisingly represses ChREBPα activity on ChREBP-specific target genes. Mechanistically, this is due to a destabilized LXRα:ChREBPα interaction, leading to reduced ChREBP-binding to chromatin and restricted activation of glycolytic and lipogenic target genes. This ligand-driven molecular switch highlights an unappreciated role of LXRα in responding to nutritional cues that was overlooked due to LXR lipogenesis-promoting function.

BackgroundVarious altitude training regimes, systematically used to improve oxygen carrying capacity and sports performance, have been associated with increased oxidative stress and inflammation. We investigated whether increased intake of common antioxidant-rich foods attenuates these processes.MethodsIn a randomized controlled trial, 31 elite endurance athletes (23 ± 5 years), ingested antioxidant-rich foods (n = 16), (> doubling their usual intake), or eucaloric control foods (n = 15) during a 3-week altitude training camp (2320 m). Fasting blood and urine samples were collected 7 days pre-altitude, after 5 and 18 days at altitude, and 7 days post-altitude. Change over time was compared between the groups using mixed models for antioxidant capacity [uric acid-free (ferric reducing ability of plasma (FRAP)], oxidative stress (8-epi-PGF2α) and inflammatory biomarkers (IFNγ, IL1α, IL1RA, IL1β, IL2, IL5, IL6, IL7, IL10, IL12p70, IL13, IL17, TNFα, MCP-1 and micro-CRP). The cytokine response to a stress-test (VO2max ramp test or 100 m swimming) was assessed at pre- and post-altitude.ResultsFRAP increased more in the antioxidant compared to the control group (p = 0.034). IL13 decreased in the antioxidant group, while increasing in the controls (p = 0.006). A similar trend was seen for IL6 (p = 0.062). A larger decrease in micro-CRP was detected in the antioxidant group compared to controls (β: -0.62, p = 0.02). We found no group differences for the remaining cytokines. 8-epi-PGF2α increased significantly in the whole population (p = 0.033), regardless group allocation. The stress response was significantly larger post-altitude compared with pre-altitude for IL1β, IL6, IL7, IL13, IL12p70 and TNFα, but we found no group differences.ConclusionsIncreased intake of antioxidant-rich foods elevated the antioxidant capacity and attenuated some of the altitude-induced systemic inflammatory biomarkers in elite athletes. The antioxidant intervention had no impact on the altitude-induced oxidative stress or changes in acute cytokine responses to exercise stress-tests.

Liver X receptor (LXR)α and LXRβ play key roles in hepatic de novo lipogenesis through their regulation of lipogenic genes, including sterol regulatory element-binding protein (SREBP)-1c and carbohydrate responsive element-binding protein (ChREBP). LXRs activate lipogenic gene transcription in response to feeding, which is believed to be mediated by insulin. We have previously shown that LXRs are targets for glucose-hexosamine-derived O-linked β-N-acetylglucosamine (O-GlcNAc) modification enhancing their ability to regulate SREBP-1c promoter activity in vitro. To elucidate insulin-independent effects of feeding on LXR-mediated lipogenic gene expression in vivo, we subjected control and streptozotocin-treated LXRα/β+/+ and LXRα/β−/− mice to a fasting-refeeding regime. We show that under hyperglycemic and hypoinsulinemic conditions, LXRs maintain their ability to upregulate the expression of glycolytic and lipogenic enzymes, including glucokinase (GK), SREBP-1c, ChREBPα, and the newly identified shorter isoform ChREBPβ. Furthermore, glucose-dependent increases in LXR/retinoid X receptor-regulated luciferase activity driven by the ChREBPα promoter was mediated, at least in part, by O-GlcNAc transferase (OGT) signaling in Huh7 cells. Moreover, we show that LXR and OGT interact and colocalize in the nucleus and that loss of LXRs profoundly reduced nuclear O-GlcNAc signaling and ChREBPα promoter binding activity in vivo. In summary, our study provides evidence that LXRs act as nutrient and glucose metabolic sensors upstream of ChREBP by modulating GK expression, nuclear O-GlcNAc signaling, and ChREBP expression and activity.

Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.