Harnessing Oxylipins and Inflammation Modulation for Prevention and Treatment of Colorectal Cancer.

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide, ranking as the third most malignant. The incidence of CRC has been increasing with time, and it is reported that Westernized diet and lifestyle play a significant role in its higher incidence and rapid progression. The intake of high amounts of omega-6 (n - 6) PUFAs and low levels of omega-3 (n - 3) PUFAs has an important role in chronic inflammation and cancer progression, which could be associated with the increase in CRC prevalence. Oxylipins generated from PUFAs are bioactive lipid mediators and have various functions, especially in inflammation and proliferation. Carcinogenesis is often a consequence of chronic inflammation, and evidence has shown the particular involvement of n - 6 PUFA arachidonic acid-derived oxylipins in CRC, which is further described in this review. A deeper understanding of the role and metabolism of PUFAs by their modifying enzymes, their pathways, and the corresponding oxylipins may allow us to identify new approaches to employ oxylipin-associated immunomodulation to enhance immunotherapy in cancer. This paper summarizes oxylipins identified in the context of the initiation, development, and metastasis of CRC. We further explore CRC chemo-prevention strategies that involve oxylipins as potential therapeutics.

Changing from lipoprotein apheresis to evolocumab treatment lowers circulating levels of arachidonic acid and oxylipins.

Background and aims: Previous studies have shown that lipoprotein apheresis can modify the plasma lipidome and pro-inflammatory and pro-thrombotic lipid mediators. This has not been examined for treatment with protein convertase subtilisin/kexin type 9 inhibitors such as evolocumab, which are increasingly used instead of lipoprotein apheresis in treatment-resistant familial hypercholesterolemia. The aim of this study was to compare the effects of evolocumab treatment and lipoprotein apheresis on the fatty acid profile and on formation of lipid mediators in blood samples. Methods: We analyzed blood samples from 37 patients receiving either lipoprotein apheresis or evolocumab treatment as part of a previous study. Patients were stratified according to receiving lipoprotein apheresis (n = 19) and evolocumab treatment (n = 18). Serum fatty acid analysis was performed using gas chromatography flame ionization detection and plasma oxylipin analysis was done using liquid chromatography tandem mass spectrometry. Results: Changing from lipoprotein apheresis to evolocumab treatment led to lower levels of omega-6 polyunsaturated fatty acid (n-6 PUFA) including arachidonic acid, dihomo-γ-linolenic acid and linoleic acid. Moreover, several n-6 PUFA-derived oxylipins were reduced after evolocumab treatment. Conclusions: Given that arachidonic acid, either directly or as a precursor, is associated with the development of inflammation and atherosclerosis, evolocumab-mediated reductions of arachidonic acid and its metabolites might have an additional beneficial effect to lower cardiovascular risk.

Ketogenic Diet Has Moderate Effects on the Fecal Microbiota of Wild-Type Mice.

The ketogenic diet (KD) is a high-fat, low-carbohydrate diet that has been reported to have neuroprotective effects. The health effects of KD might be linked to an altered gut microbiome, which plays a major role in host health, leading to neuroprotective effects via the gut-brain axis. However, results from different studies, most often based on the 16S rRNA gene and metagenome sequencing, have been inconsistent. In this study, we assessed the effect of a 4-week KD compared to a western diet (WD) on the colonic microbiome of female C57Bl/6J mice by analyzing fecal samples using fluorescence in situ hybridization. Our results showed distinct changes in the total number of gut bacteria following the 4-week KD, in addition to changes in the composition of the microbiome. KD-fed mice showed higher absolute numbers of Actinobacteria (especially Bifidobacteria spp.) and lower absolute levels of Proteobacteria, often linked to gut inflammation, in comparison with WD-fed mice. Furthermore, an increased abundance of the typically rare genus Atopobium was observed. These changes may indicate the possible anti-inflammatory effects of the KD. However, since the overall changes in the microbiota seem low, the KD effects might be linked to the differential abundance of only a few key genera in mice.

Bioactive oxylipins in type 2 diabetes mellitus patients with and without hypertriglyceridemia.

Objective: Dyslipidemia, in particular elevated triglycerides (TGs) contribute to increased cardiovascular risk in type 2 diabetes mellitus (T2DM). In this pilot study we aimed to assess how increased TGs affect hepatic fat as well as polyunsaturated fatty acid (PUFA) metabolism and oxylipin formation in T2DM patients. Methods: 40 patients with T2DM were characterized analyzing routine lipid blood parameters, as well as medical history and clinical characteristics. Patients were divided into a hypertriglyceridemia (HTG) group (TG ≥ 1.7mmol/l) and a normal TG group with TGs within the reference range (TG < 1.7mmol/l). Profiles of PUFAs and their oxylipins in plasma were measured by gas chromatography and liquid chromatography/tandem mass spectrometry. Transient elastography (TE) was used to assess hepatic fat content measured as controlled attenuation parameter (CAP) (in dB/m) and the degree of liver fibrosis measured as stiffness (in kPa). Results: Mean value of hepatic fat content measured as CAP as well as body mass index (BMI) were significantly higher in patients with high TGs as compared to those with normal TGs, and correlation analysis showed higher concentrations of TGs with increasing CAP and BMI scores in patients with T2DM. There were profound differences in plasma oxylipin levels between these two groups. Cytochrome P450 (CYP) and lipoxygenase (LOX) metabolites were generally more abundant in the HTG group, especially those derived from arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), γ-linolenic acid (γ-LA), and α-linolenic acid (α-LA), and a strong correlation between TG levels and plasma metabolites from different pathways was observed. Conclusions: In adult patients with T2DM, elevated TGs were associated with increased liver fat and BMI. Furthermore, these patients also had significantly higher plasma levels of CYP- and LOX- oxylipins, which could be a novel indicator of increased inflammatory pathway activity, as well as a novel target to dampen this activity.

Transgenic mice overexpressing human ALOX15 under the control of the aP2 promoter are partly protected in the complete Freund's adjuvant-induced paw inflammation model.

BACKGROUND, OBJECTIVES AND DESIGN: Arachidonic acid 15-lipoxygenase (ALOX15) has been implicated in the pathogenesis of inflammatory diseases but since pro- and anti-inflammatory roles have been suggested, the precise function of this enzyme is still a matter of discussion. To contribute to this discussion, we created transgenic mice, which express human ALOX15 under the control of the activating protein 2 promoter (aP2-ALOX15 mice) and compared the sensitivity of these gain-of-function animals in two independent mouse inflammation models with Alox15-deficient mice (loss-of-function animals) and wildtype control animals. MATERIALS AND METHODS: Transgenic aP2-ALOX15 mice were tested in comparison with Alox15 knockout mice (Alox15-/-) and corresponding wildtype control animals (C57BL/6J) in the complete Freund's adjuvant induced hind-paw edema model and in the dextran sulfate sodium induced colitis (DSS-colitis) model. In the paw edema model, the degree of paw swelling and the sensitivity of the inflamed hind-paw for mechanic (von Frey test) and thermal (Hargreaves test) stimulation were quantified as clinical readout parameters. In the dextran sodium sulfate induced colitis model the loss of body weight, the colon lengths and the disease activity index were determined. RESULTS: In the hind-paw edema model, systemic inactivation of the endogenous Alox15 gene intensified the inflammatory symptoms, whereas overexpression of human ALOX15 reduced the degree of hind-paw inflammation. These data suggest anti-inflammatory roles for endogenous and transgenic ALOX15 in this particular inflammation model. As mechanistic reason for the protective effect downregulation of the pro-inflammatory ALOX5 pathways was suggested. However, in the dextran sodium sulfate colitis model, in which systemic inactivation of the Alox15 gene protected female mice from DSS-induced colitis, transgenic overexpression of human ALOX15 did hardly impact the intensity of the inflammatory symptoms. CONCLUSION: The biological role of ALOX15 in the pathogenesis of inflammation is variable and depends on the kind of the animal inflammation model.

Prevention of colitis-induced liver oxidative stress and inflammation in a transgenic mouse model with increased omega-3 polyunsaturated fatty acids.

Inflammatory bowel disease (IBD) is an immune-mediated gut dysfunction, which might also be associated with an inflammatory phenotype in the liver. It is known that the nutritional intake of omega-3 polyunsaturated fatty acids (n-3 PUFA) is inversely correlated to the severity and occurrence of IBD. In order to investigate whether n-3 PUFA can also reduce liver inflammation and oxidative liver damage due to colon inflammation, we explored the dextran sulfate sodium (DSS)-induced colitis model in wild-type and fat-1 mice with endogenously increased n-3 PUFA tissue content. Besides confirming previous data of alleviated DSS-induced colitis in the fat-1 mouse model, the increase of n-3 PUFA also resulted in a significant reduction of liver inflammation and oxidative damage in colitis-affected fat-1 mice as compared to wild-type littermates. This was accompanied by a remarkable increase of established inflammation-dampening n-3 PUFA oxylipins, namely docosahexaenoic acid-derived 19,20-epoxydocosapentaenoic acid and eicosapentaenoic acid-derived 15-hydroxyeicosapentaenoic acid and 17,18-epoxyeicosatetraenoic acid. Taken together, these observations demonstrate a strong inverse correlation between the anti-inflammatory lipidome derived from n-3 PUFA and the colitis-triggered inflammatory changes in the liver by reducing oxidative liver stress.

Sorafenib increases cytochrome P450 lipid metabolites in patient with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death, and medical treatment options are limited. The multikinase inhibitor sorafenib was the first approved drug widely used for systemic therapy in advanced HCC. Sorafenib might affect polyunsaturated fatty acids (PUFA)-derived epoxygenated metabolite levels, as it is also a potent inhibitor of the soluble epoxide hydrolase (sEH), which catalyzes the conversion of cytochrome-P450 (CYP)-derived epoxide metabolites derived from PUFA, such as omega-6 arachidonic acid (AA) and omega-3 docosahexaenoic acid (DHA), into their corresponding dihydroxy metabolites. Experimental studies with AA-derived epoxyeicosatrienoic acids (EETs) have shown that they can promote tumor growth and metastasis, while DHA-derived 19,20-epoxydocosapentaenoic acid (19,20-EDP) was shown to have anti-tumor activity in mice. In this study, we found a significant increase in EET levels in 43 HCC patients treated with sorafenib and a trend towards increased levels of DHA-derived 19,20-EDP. We demonstrate that the effect of sorafenib on CYP- metabolites led to an increase of 19,20-EDP and its dihydroxy metabolite, whereas DHA plasma levels decreased under sorafenib treatment. These data indicate that specific supplementation with DHA could be used to increase levels of the epoxy compound 19,20-EDP with potential anti-tumor activity in HCC patients receiving sorafenib therapy.

Corrigendum: Sorafenib increases cytochrome P450 lipid metabolites in patient with hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fphar.2023.1124214.].

Formation, Signaling and Occurrence of Specialized Pro-Resolving Lipid Mediators-What is the Evidence so far?

Formation of specialized pro-resolving lipid mediators (SPMs) such as lipoxins or resolvins usually involves arachidonic acid 5-lipoxygenase (5-LO, ALOX5) and different types of arachidonic acid 12- and 15-lipoxygenating paralogues (15-LO1, ALOX15; 15-LO2, ALOX15B; 12-LO, ALOX12). Typically, SPMs are thought to be formed via consecutive steps of oxidation of polyenoic fatty acids such as arachidonic acid, eicosapentaenoic acid or docosahexaenoic acid. One hallmark of SPM formation is that reported levels of these lipid mediators are much lower than typical pro-inflammatory mediators including the monohydroxylated fatty acid derivatives (e.g., 5-HETE), leukotrienes or certain cyclooxygenase-derived prostaglandins. Thus, reliable detection and quantification of these metabolites is challenging. This paper is aimed at critically evaluating i) the proposed biosynthetic pathways of SPM formation, ii) the current knowledge on SPM receptors and their signaling cascades and iii) the analytical methods used to quantify these pro-resolving mediators in the context of their instability and their low concentrations. Based on current literature it can be concluded that i) there is at most, a low biosynthetic capacity for SPMs in human leukocytes. ii) The identity and the signaling of the proposed G-protein-coupled SPM receptors have not been supported by studies in knock-out mice and remain to be validated. iii) In humans, SPM levels were neither related to dietary supplementation with their ω-3 polyunsaturated fatty acid precursors nor were they formed during the resolution phase of an evoked inflammatory response. iv) The reported low SPM levels cannot be reliably quantified by means of the most commonly reported methodology. Overall, these questions regarding formation, signaling and occurrence of SPMs challenge their role as endogenous mediators of the resolution of inflammation.

Knock-In Mice Expressing a 15-Lipoxygenating Alox5 Mutant Respond Differently to Experimental Inflammation Than Reported Alox5-/- Mice.

Arachidonic acid 5-lipoxygenase (ALOX5) is the key enzyme in the biosynthesis of pro-inflammatory leukotrienes. We recently created knock-in mice (Alox5-KI) which express an arachidonic acid 15-lipoxygenating Alox5 mutant instead of the 5-lipoxygenating wildtype enzyme. These mice were leukotriene deficient but exhibited an elevated linoleic acid oxygenase activity. Here we characterized the polyenoic fatty acid metabolism of these mice in more detail and tested the animals in three different experimental inflammation models. In experimental autoimmune encephalomyelitis (EAE), Alox5-KI mice displayed an earlier disease onset and a significantly higher cumulative incidence rate than wildtype controls but the clinical score kinetics were not significantly different. In dextran sodium sulfate-induced colitis (DSS) and in the chronic constriction nerve injury model (CCI), Alox5-KI mice performed like wildtype controls with similar genetic background. These results were somewhat surprising since in previous loss-of-function studies targeting leukotriene biosynthesis (Alox5-/- mice, inhibitor studies), more severe inflammatory symptoms were observed in the EAE model but the degree of inflammation in DSS colitis was attenuated. Taken together, our data indicate that these mutant Alox5-KI mice respond differently in two models of experimental inflammation than Alox5-/- animals tested previously in similar experimental setups.

Omega-3 fatty acids protect from colitis via an Alox15-derived eicosanoid.

An increased omega-3 polyunsaturated fatty acid (n-3 PUFA) tissue status can lead to a significant formation of anti-inflammatory lipid mediators and effective reduction in inflammation and tissue injury in murine colitis. Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory bowel disease as well as in the formation of pro- and anti-inflammatory lipid mediators. To explore the role of Alox15 in the protective response found in fat1 transgenic mice with endogenously increased n-3 PUFA tissue status fat1 transgenic mice were crossed with Alox15-deficient animals and challenged in the dextran sulfate sodium (DSS)- and the 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis model. Transgenic fat1 mice rich in endogenous n-3 PUFAs were protected from colitis. However, additional systemic inactivation of the Alox15 gene counteracted this protective effect. To explore the molecular basis for this effect Alox15 lipid metabolites derived from n-3 PUFA were analyzed in the different mice. Alox15 deficiency suppressed the formation of n-3 PUFA-derived 15-hydroxy eicosapentaenoic acid (15-HEPE). In contrast, treating mice with intraperitoneal injections of 15S-HEPE protected wild-type mice from DSS- and TNBS-induced colitis. These data suggest that the anti-colitis effect of increased n-3 PUFA in the transgenic fat1 mouse model is mediated in part via Alox15-derived 15-HEPE formation.

Effects of chronic low-dose aspirin treatment on tumor prevention in three mouse models of intestinal tumorigenesis.

Although early detection and treatment of colorectal cancer (CRC) have improved, it remains a significant health-care problem with high morbidity and mortality. Data indicate that long-term intake of low-dose aspirin reduces the risk of CRC; however, the mechanisms underlying this chemopreventive effect are still unclear. Different mouse models for inflammation-associated, sporadic, and hereditary CRC were applied to assess the efficacy and mechanism of low-dose aspirin on tumor prevention. An initial dosing study performed in healthy mice indicates that aspirin at a dose of 25 mg/kg/d has a similar pharmacodynamic effect as low-dose aspirin treatment in human subjects (100 mg/d). Chronic low-dose aspirin treatment suppresses colitis-associated and to a lesser extent spontaneous tumorigenesis in mice. Aspirin's antitumor effect is most pronounced in a preventive approach when aspirin administration starts before the tumor-initiating genotoxic event and continues for the duration of the experiment. These effects are not associated with alterations in cell proliferation, apoptosis, or activation of signaling pathways involved in CRC. Aspirin-induced reduction in tumor burden is accompanied by inhibition of thromboxane B2 formation, indicating reduced platelet activation. Aspirin treatment also results in decreased colonic prostaglandin E2 formation and tumor angiogenesis. With respect to colitis-triggered tumorigenesis, aspirin administration is associated with a reduction in inflammatory activity in the colon, as indicated by decreased levels of pro-inflammatory mediators, and tumor-associated iNOS-positive macrophages. Our results suggest that low-dose aspirin represents an effective antitumor agent in the context of colon tumorigenesis primarily due to its well-established cyclooxygenase inhibition effects.

Non-canonical HIF-1 stabilization contributes to intestinal tumorigenesis.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1's role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of Wnt/ß-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts (TAF) and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with HIF-1α, arguing for an importance of hypoxia-independent, i.e., non-canonical, HIF-1 stabilization for intestinal tumorigenesis that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1α stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

Female mice carrying a defective Alox15 gene are protected from experimental colitis via sustained maintenance of the intestinal epithelial barrier function.

Lipoxygenases (ALOXs) are involved in the regulation of cellular redox homeostasis. They also have been implicated in the biosynthesis of pro- and anti-inflammatory lipid mediators and play a role in the pathogenesis of inflammatory diseases, which constitute a major health challenge owing to increasing incidence and prevalence in all industrialized countries around the world. To explore the pathophysiological role of Alox15 (leukocyte-type 12-LOX) in mouse experimental colitis we tested the impact of systemic inactivation of the Alox15 gene on the extent of dextrane sulfate sodium (DSS) colitis. We found that in wildtype mice expression of the Alox15 gene was augmented during DSS-colitis while expression of other Alox genes (Alox5, Alox15b) was hardly altered. Systemic Alox15 (leukocyte-type 12-LOX) deficiency induced less severe colitis symptoms and suppressed in vivo formation of 12-hydroxyeicosatetraenoic acid (12-HETE), the major Alox15 (leukocyte-type 12-LOX) product in mice. These alterations were paralleled by reduced expression of pro-inflammatory gene products, by sustained expression of the zonula occludens protein 1 (ZO-1) and by a less impaired intestinal epithelial barrier function. These results are consistent with in vitro incubations of colon epithelial cells, in which addition of 12S-HETE compromised enantioselectively transepithelial electric resistance. Consistent with these data transgenic overexpression of human ALOX15 intensified the inflammatory symptoms. In summary, our results indicate that systemic Alox15 (leukocyte-type 12-LOX) deficiency protects mice from DSS-colitis. Since exogenous 12-HETE compromises the expression of the tight junction protein ZO-1 the protective effect has been related to a less pronounced impairment of the intestinal epithelial barrier function.

Aspirin alone and combined with a statin suppresses eicosanoid formation in human colon tissue.

Eicosanoids, including prostaglandins (PGs) and thromboxanes, are broadly bioactive lipid mediators and increase colon tumorigenesis possibly through chronic inflammatory mechanisms. Epidemiological and experimental data suggest that acetylsalicylic acid (ASA) helps prevent colorectal cancer (CRC), possibly through cyclooxygenase (COX)-mediated suppression of eicosanoid, particularly PGE2, formation. Recent studies suggest that statins prevent CRC and improve survival after diagnosis. We identified patients on ASA and/or statin treatment undergoing routine colonoscopy and measured eicosanoid levels in colonic mucosa with targeted metabolomics technology (LC-MS/MS). ASA-treated individuals (n = 27) had significantly lower tissue eicosanoid levels of most COX-derived metabolites than untreated individuals (n = 31). In contrast, COX-derived lipid metabolites tended to be higher in patients with statin treatment (n = 7) as compared with those not receiving statins (n = 24). This effect was not discernible in subjects treated with ASA and statins (n = 11): Individuals treated with both drugs showed a pronounced suppression of COX-derived eicosanoids in colon tissue, even compared with subjects treated with ASA alone. Our data from a routine clinical setting support the hypothesis that ASA and statins could inhibit CRC development via lipid mediator modification. Further studies should directly investigate the effect of dual ASA and statin treatment on colon tumorigenesis in humans.

Autofluorescence: A potential pitfall in immunofluorescence-based inflammation grading.

Immunofluorescence (IF) staining of paraffin-embedded tissues is a frequently used method to answer research questions or even detect the abundance of a certain protein for diagnostic use. However, the signal originating from specific antibody-staining might be distorted by autofluorescence (AF) of the assessed tissue. Although the AF phenomenon is well known, its presence is often neglected by insufficient staining controls. In this study, we describe the existence of cellular AF in paraffin-embedded healthy and inflamed human and murine colonic tissues and present ways to reduce AF. The AF signal is detectable at emission spectra from 425 nm-738 nm, upon excitation from 403.6-638.7 nm and appears more pronounced in inflamed tissues. Most signals are located subepithelially in the tissue and in blood vessels. Previous studies have shown that the AF signals are caused by lipofuscin, which accumulates in lamina propria immune cells. In murine small intestine AF signals are present in granules in the Paneth cell zone. For alleviation of the AF signal, sudan black b (SBB) or copper sulfate was used. Incubation of the tissue slices with either one of the substances reduced AF. In conclusion, AF appears as an intrinsic biomarker for colonic inflammation. The dominant existence of AF in immune cells of IBD tissue elucidates the importance of negative controls and the limitation of IF staining for potential diagnostic purposes.

Modulation of the endogenous omega-3 fatty acid and oxylipin profile in vivo-A comparison of the fat-1 transgenic mouse with C57BL/6 wildtype mice on an omega-3 fatty acid enriched diet.

Dietary intervention and genetic fat-1 mice are two models for the investigation of effects associated with omega-3 polyunsaturated fatty acids (n3-PUFA). In order to assess their power to modulate the fatty acid and oxylipin pattern, we thoroughly compared fat-1 and wild-type C57BL/6 mice on a sunflower oil diet with wild-type mice on the same diet enriched with 1% EPA and 1% DHA for 0, 7, 14, 30 and 45 days. Feeding led after 14-30 days to a high steady state of n3-PUFA in all tissues at the expense of n6-PUFAs. Levels of n3-PUFA achieved by feeding were higher compared to fat-1 mice, particularly for EPA (max. 1.7% in whole blood of fat-1 vs. 7.8% following feeding). Changes in PUFAs were reflected in most oxylipins in plasma, brain and colon: Compared to wild-type mice on a standard diet, arachidonic acid metabolites were overall decreased while EPA and DHA oxylipins increased with feeding more than in fat-1 mice. In plasma of n3-PUFA fed animals, EPA and DHA metabolites from the lipoxygenase and cytochrome P450 pathways dominated over ARA derived counterparts.Fat-1 mice show n3-PUFA level which can be reached by dietary interventions, supporting the applicability of this model in n3-PUFA research. However, for specific questions, e.g. the role of EPA derived mediators or concentration dependent effects of (individual) PUFA, feeding studies are necessary.

Annexin A1 sustains tumor metabolism and cellular proliferation upon stable loss of HIF1A.

Despite the approval of numerous molecular targeted drugs, long-term antiproliferative efficacy is rarely achieved and therapy resistance remains a central obstacle of cancer care. Combined inhibition of multiple cancer-driving pathways promises to improve antiproliferative efficacy. HIF-1 is a driver of gastric cancer and considered to be an attractive target for therapy. We noted that gastric cancer cells are able to functionally compensate the stable loss of HIF-1α. Via transcriptomics we identified a group of upregulated genes in HIF-1α-deficient cells and hypothesized that these genes confer survival upon HIF-1α loss. Strikingly, simultaneous knock-down of HIF-1α and Annexin A1 (ANXA1), one of the identified genes, resulted in complete cessation of proliferation. Using stable isotope-resolved metabolomics, oxidative and reductive glutamine metabolism was found to be significantly impaired in HIF-1α/ANXA1-deficient cells, potentially explaining the proliferation defect. In summary, we present a conceptually novel application of stable gene inactivation enabling in-depth deconstruction of resistance mechanisms. In theory, this experimental approach is applicable to any cancer-driving gene or pathway and promises to identify various new targets for combination therapies.

Time course of contrast enhancement by micro-CT with dedicated contrast agents in normal mice and mice with hepatocellular carcinoma: comparison of one iodinated and two nanoparticle-based agents.

RATIONALE AND OBJECTIVES: The aim of the present study was to characterize the kinetics of two nanoparticle-based contrast agents for preclinical imaging, Exitron nano 6000 and Exitron nano 12000, and the iodinated agent eXIA 160 in both healthy mice and in a mouse model of hepatocellular carcinoma (HCC). Semiautomatic segmentation of liver lesions for estimation of total tumor load of the liver was evaluated in HCC mice. MATERIALS AND METHODS: The normal time course of contrast enhancement was assessed in 15 healthy C57BL/6 mice. Imaging of tumor spread in the liver was evaluated in 15 mice harboring a transgenic HCC model (ASV-B mice). Automatic segmentation of liver lesions for determination of total tumor burden of the liver was tested in three additional ASV-B mice before and after an experimental therapy. RESULTS: In healthy mice, clearance of the contrast agent from blood was completed within 3-4 hours for eXIA 160 and Exitron nano 6000, whereas complete blood clearance of Exitron nano 12000 required about 24 hours. eXIA 160 provided maximum liver contrast at 1 hour post injection (p.i.) followed by a continuous decline. Enhancement of liver contrast with Exitron nano 6000 and Exitron nano 12000 reached a plateau at about 4 hours p.i., which lasted until the end of the measurements at 96 hours p.i. Maximum contrast enhancement of the liver was not statistically different between Exitron nano 6000 and Exitron nano 12000, but was about three times lower for eXIA 160 (P < .05). Visually Exitron nano 12000 provided the best liver-to-tumor contrast. Semiautomatic liver and tumor segmentation was feasible after the administration of Exitron nano 12000 but did not work properly for the other two contrast agents. CONCLUSIONS: Both nanoparticle-based contrast agents provided stronger and longer lasting contrast enhancement of healthy liver parenchyma. Exitron nano 12000 allowed automatic segmentation of tumor lesions for estimation of the total tumor load in the liver.

HIF1α modulates cell fate reprogramming through early glycolytic shift and upregulation of PDK1-3 and PKM2.

Reprogramming somatic cells to a pluripotent state drastically reconfigures the cellular anabolic requirements, thus potentially inducing cancer-like metabolic transformation. Accordingly, we and others previously showed that somatic mitochondria and bioenergetics are extensively remodeled upon derivation of induced pluripotent stem cells (iPSCs), as the cells transit from oxidative to glycolytic metabolism. In the attempt to identify possible regulatory mechanisms underlying this metabolic restructuring, we investigated the contributing role of hypoxia-inducible factor one alpha (HIF1α), a master regulator of energy metabolism, in the induction and maintenance of pluripotency. We discovered that the ablation of HIF1α function in dermal fibroblasts dramatically hampers reprogramming efficiency, while small molecule-based activation of HIF1α significantly improves cell fate conversion. Transcriptional and bioenergetic analysis during reprogramming initiation indicated that the transduction of the four factors is sufficient to upregulate the HIF1α target pyruvate dehydrogenase kinase (PDK) one and set in motion the glycolytic shift. However, additional HIF1α activation appears critical in the early upregulation of other HIF1α-associated metabolic regulators, including PDK3 and pyruvate kinase (PK) isoform M2 (PKM2), resulting in increased glycolysis and enhanced reprogramming. Accordingly, elevated levels of PDK1, PDK3, and PKM2 and reduced PK activity could be observed in iPSCs and human embryonic stem cells in the undifferentiated state. Overall, the findings suggest that the early induction of HIF1α targets may be instrumental in iPSC derivation via the activation of a glycolytic program. These findings implicate the HIF1α pathway as an enabling regulator of cellular reprogramming.