Epigenetic priming targets tumor heterogeneity to shift transcriptomic phenotype of pancreatic ductal adenocarcinoma towards a Vitamin D susceptible state.

As a highly heterogeneous tumor, pancreatic ductal adenocarcinoma (PDAC) exhibits non-uniform responses to therapies across subtypes. Overcoming therapeutic resistance stemming from this heterogeneity remains a significant challenge. Here, we report that Vitamin D-resistant PDAC cells hijacked Vitamin D signaling to promote tumor progression, whereas epigenetic priming with glyceryl triacetate (GTA) and 5-Aza-2'-deoxycytidine (5-Aza) overcame Vitamin D resistance and shifted the transcriptomic phenotype of PDAC toward a Vitamin D-susceptible state. Increasing overall H3K27 acetylation with GTA and reducing overall DNA methylation with 5-Aza not only elevated the Vitamin D receptor (VDR) expression but also reprogrammed the Vitamin D-responsive genes. Consequently, Vitamin D inhibited cell viability and migration in the epigenetically primed PDAC cells by activating genes involved in apoptosis as well as genes involved in negative regulation of cell proliferation and migration, while the opposite effect of Vitamin D was observed in unprimed cells. Studies in genetically engineered mouse PDAC cells further validated the effects of epigenetic priming for enhancing the anti-tumor activity of Vitamin D. Using gain- and loss-of-function experiments, we further demonstrated that VDR expression was necessary but not sufficient for activating the favorable transcriptomic phenotype in respond to Vitamin D treatment in PDAC, highlighting that both the VDR and Vitamin D-responsive genes were prerequisites for Vitamin D response. These data reveal a previously undefined mechanism in which epigenetic state orchestrates the expression of both VDR and Vitamin D-responsive genes and determines the therapeutic response to Vitamin D in PDAC.

Urine 5-Eicosatetraenoic Acids as Diagnostic Markers for Obstructive Sleep Apnea.

Early detection of obstructive sleep apnea (OSA) is needed to reduce cardiovascular sequelae and mortality. Full-night polysomnography has been used for diagnosing OSA, but it is too expensive and inconvenient for patients to handle. Metabolome-wide analyses were performed to find and validate surrogate markers for OSA. We further investigated the mechanism underlying hypoxic induction of the markers in human cells and mice. Arachidonic acid derivatives 5-HETE and 5-oxoETE were detected in urine samples. The levels (mean ± SD, ng per mg creatinine) of 5-HETE and 5-oxoETE were 56.4 ± 26.2 and 46.9 ± 18.4 in OSA patients, respectively, which were significantly higher than those in controls (22.5 ± 4.6 and 18.7 ± 3.6). Both levels correlated with the apnea-hypopnea index and the lowest oxygen saturation on polysomnography. After the treatment with the continuous positive airway pressure, the metabolite levels were significantly reduced compared with those before the treatment. In human mononuclear cells subjected to intermittent hypoxia, 5-HETE and 5-oxoETE productions were induced by hypoxia-inducible factor 1 and glutathione peroxidase. When mice were exposed to intermittent hypoxia, 5-HETE and 5-oxoETE were excreted more in urine. They were identified and verified as new OSA markers reflecting hypoxic stress. The OSA markers could be used for OSA diagnosis and therapeutic evaluation.

Probiotic treatment induced change of inflammation related metabolites in IBS-D patients/double-blind, randomized, placebo-controlled trial.

There have been many studies suggesting that probiotics are effective in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). However, its mechanism of action as well as prediction of response is still to be elucidated. In the present study, to find out metabolomic characteristics of probiotic effect in IBS-D, we compared IBS symptom changes and metabolomic characteristics in the subjects' urine samples between multi-strain probiotics (one strain of Lactobacillus sp. and four strains of Bifidobacterium sp.) group (n = 32) and placebo group (n = 31). After 8 weeks' administration (3 times/day), dissatisfaction in bowel habits and stool frequencies were significantly improved. Also, probiotics group had significantly changed seven metabolites including palmitic acid methyl ester (PAME) and 4,6-dihydroxyquinoline, 4-(2-aminophenyl)-2,4-dioxobutanoic acid (DOBA). According to IBS-SSS and IBS-QoL questionnaires, IBS-SSS responders showed higher PAME levels and IBS-QoL responders showed lower DOBA levels. This suggests potential role of these metabolites as a biomarker to predict probiotics effect in IBS-D patients.

Integrative Analysis of Renal Ischemia/Reperfusion Injury and Remote Ischemic Preconditioning in Mice.

Remote ischemic preconditioning (RIPC) is a strategy to induce resistance in a target organ against the oxidative stress and injury caused by ischemia and reperfusion (IR). RIPC harnesses the body's endogenous protective capabilities through brief episodes of IR applied in organs remote from the target. Few studies have analyzed this phenomenon in the kidney. Furthermore, the window of protection representing RIPC efficacy has not been fully elucidated. Here, we performed a multiomics study to specify those associated with protective effects of RIPC against the IR injury. A total of 30 mice were divided to four groups: sham, IR only, late RIPC + IR, and early RIPC + IR. We found that IR clearly led to tubular injury, whereas both preconditioning groups exhibited attenuated injury after the insult. In addition, renal IR injury produced changes of the metabolome in kidney, serum, and urine specimens. Furthermore, distinctive mRNA and associated protein expression changes supported potential mechanisms. Our findings revealed that RIPC effectively reduces renal damage after IR and that the potential mechanisms differed between the two time windows of protection. These results may potentially be extended to humans to allow non- or minimally invasive diagnosis of renal IR injury and RIPC efficacy.

Plasma LTE4/PGF2α Ratio and Blood Eosinophil Count Are Increased in Elderly Asthmatics With Previous Asthma Exacerbation.

The tools for asthma control assessment recommended by the current guideline are cognitive function- and effort-dependent, which is substantially impaired in the elderly. The aim of this study is to investigate objective assessment tools of asthma control status and previous asthma exacerbation (AE) in elderly subjects. Asthmatics aged >60 years who were treated with step 2 or 3 by the Global Initiative for Asthma (GINA) guideline were enrolled. During the 12-week study period, the subjects used either 400 µg of budesonide plus 10 mg of montelukast or 800 µg of inhaled budesonide. The occurrence of AE during the 4-week run-in and 12-week treatment period was monitored. After 12-week of treatment, sputum eosinophil count, peripheral eosinophil count, the plasma leukotriene E4 (LTE4), and prostaglandin F2α (PGF2α) metabolite levels were measured using the UHPLC/Q-ToF MS system. The study subjects were divided into group 1 (asthmatics who experienced AE during the study period) and group 2 (those who did not). A total of 101 patients aged 60-85 years were enrolled. Twenty-three patients (22.8%) had experienced AE. The plasma LTE4 level, LTE4/PGF2α ratio, and peripheral eosinophil count were significantly higher in group 1 than in group 2 (P=0.023, P=0.010, P=0.033, respectively). The plasma LTE4/PGF2α ratio and peripheral eosinophil count at week 12 were significantly associated with previous AE (odds ratio [OR]=1.748, P=0.013; OR=1.256, P=0.027). Receiver operating characteristic (ROC) curves to discriminate the subjects with previous AE, including these 2 parameters, showed that the area under the curve was 0.700 (P=0.004), with 73.9% sensitivity and 47.9% specificity. In conclusion, a combination of plasma LTE4/PGF2α ratio and peripheral eosinophil count can be an objective assessment tool which is significantly associated with asthma control status in elderly asthmatics.

Exploration of the Sphingolipid Metabolite, Sphingosine-1-phosphate and Sphingosine, as Novel Biomarkers for Aspirin-exacerbated Respiratory Disease.

Sphingolipid (SL) metabolites have been suggested to be important inflammatory mediators in airway inflammation and asthma. However, little is known about SL metabolites in aspirin-exacerbated respiratory disease (AERD). We aimed to explore the potential AERD biomarkers by conducting lipidomics targeting SL metabolites. The levels of SL metabolites in serum and urine samples from 45 AERD patients and 45 aspirin-tolerant asthma (ATA) patients were quantified through mass spectrometry. During the lysine-aspirin bronchoprovocation test (ASA-BPT), the levels of serum sphingomyelin (SM) were significantly decreased in AERD (P < 0.05) but not in ATA. The serum SM levels were positively correlated with airway responsiveness to methacholine. At the basal status before the ASA-BPT, the levels of serum sphingosine-1-phosphate (S1P) and urine sphingosine were significantly higher in the AERD patients compared with that of ATA patients (P < 0.001) and were positively correlated with a greater decrease in FEV1 (%) values following the ASA-BPT test (P < 0.001 for each), and with serum periostin level (P < 0.05 for each). This study is the first to evaluate serum S1P and urine sphingosine as potential biomarkers of AERD as well as to examine the metabolic disturbance of SL in AERD patients.

Antihyperglycemic mechanism of metformin occurs via the AMPK/LXRα/POMC pathway.

Metformin is a first-line drug for treating type 2 diabetes. Although metformin is known to phosphorylate AMP-activated protein kinase (AMPK), it is unclear how the glucose-lowering effect of metformin is related to AMPK activation. The aim of this study was to identify the urinary endogenous metabolites affected by metformin and to identify the novel underlying molecular mechanisms related to its anti-diabetic effect. Fourteen healthy male subjects were orally administered metformin (1000 mg) once. First morning urine samples were taken before and after administration to obtain metabolomic data. We then further investigated the anti-diabetic mechanism of metformin in vitro and in vivo. The fluctuation of the metabolite cortisol indicated that the neuroendocrine system was involved in the anti-diabetic effect of metformin. Actually we found that metformin induced AMPK/liver X receptor α (LXRα) phosphorylation, followed by pro-opiomelanocortin (POMC) suppression in rat pituitary cells. We confirmed this result by administering metformin in an animal study. Given that cortisol stimulates gluconeogenesis, we propose the anti-hyperglycemic effect of metformin is attributed to reduced POMC/adrenocorticotropic hormone (ACTH)/cortisol levels following AMPK/LXRα phosphorylation in the pituitaries.

ITF2 prevents activation of the ß-catenin-TCF4 complex in colon cancer cells and levels decrease with tumor progression.

BACKGROUND & AIMS: Immunoglobulin transcription factor 2 (ITF2) was believed to promote neoplastic transformation via activation of ß-catenin. However, ITF2 recently was reported to suppress colon carcinogenesis. We investigated the roles of ITF2 in colorectal cancer cell lines and tumor formation and growth in mice. METHODS: Levels of ITF2, ß-catenin, and c-Myc were measured in 12 human colorectal tumor samples and by immunohistochemistry. ITF2 regulation of ß-catenin and T-cell factor (TCF) were analyzed using luciferase reporter, reverse-transcription quantitative polymerase chain reaction, flow cytometry, and immunoblot analyses. Mice were given subcutaneous injections of human colorectal cancer cell lines that stably express ITF2, small hairpin RNAs to reduce levels of ITF2, or control plasmids; xenograft tumor growth was assessed. Human colorectal carcinoma tissue arrays were used to associate levels of ITF2 expression and clinical outcomes. RESULTS: Levels of ß-catenin, cMyc, and ITF2 were increased in areas of human colon adenomas and carcinomas, compared with nontumor areas of the same tissues. ITF2 levels were reduced and cMyc levels were increased in areas of carcinoma, compared with adenoma. In human colorectal cancer cell lines, activation of the ß-catenin-TCF4 complex and expression of its target genes were regulated negatively by ITF2. ITF2 inhibited formation of the ß-catenin-TCF4 complex by competing with TCF4 for ß-catenin binding. Stable transgenic expression of ITF2 in human colorectal cancer cell lines reduced their proliferation and tumorigenic potential in mice, whereas small hairpin RNA knockdown of ITF2 promoted growth of xenograft tumors in mice. In an analysis of colorectal tumor tissue arrays, loss of ITF2 from colorectal tumor tissues was associated with poor outcomes of patients. A gene set enrichment analysis supported the negative correlation between the level of ITF2 and activity of the ß-catenin-TCF4 complex. CONCLUSIONS: In human colorectal cancer cell lines and tissue samples, ITF2 appears to prevent activation of the ß-catenin-TCF4 complex and transcription of its gene targets. Loss of ITF2 promotes the ability of colorectal cancer cells to form xenograft tumors, and is associated with tumor progression and shorter survival times of patients.

Disulfiram deregulates HIF-α subunits and blunts tumor adaptation to hypoxia in hepatoma cells.

AIM: Disulfiram is an aldehyde dehydrogenase inhibitor that was used to treat alcoholism and showed anticancer activity, but its anticancer mechanism remains unclear. The aim of this study was to investigate the effects of disulfiram on the hypoxia-inducible factor (HIF)-driven tumor adaptation to hypoxia in vitro. METHODS: Hep3B, Huh7 and HepG2 hepatoma cells were incubated under normoxic (20% O2) or hypoxic (1% O2) conditions for 16 h. The expression and activity of HIF-1α and HIF-2α proteins were evaluated using immunoblotting and luciferase reporter assay, respectively. Semi-quantitative RT-PCR was used to analyze HIF-mediated gene expression. Endothelial tubule formation assay was used to evaluate the anti-angiogenic effect. RESULTS: Hypoxia caused marked expression of HIF-1α and HIF-1α in the 3 hepatoma cell lines, dramatically increased HIF activity and induced the expression of HIF downstream genes (EPO, CA9, VEGF-A and PDK1) in Hep3B cells. HIF-2α expression was positively correlated with the induction of hypoxic genes (CA9, VEGF-A and PDK1). Moreover, hypoxia markedly increased VEGF production and angiogenic potential of Hep3B cells. Disulfiram (0.3 to 2 µmol/L) inhibited hypoxia-induced gene expression and HIF activity in a dose-dependent manner. Disulfiram more effectively suppressed the viability of Hep3B cells under hypoxia, but it did not affect the cell cycle. Overexpression of HIF-2α in Hep3B cells reversed the inhibitory effects of disulfiram on hypoxia-induced gene expression and cell survival under hypoxia. CONCLUSION: Disulfiram deregulates the HIF-mediated hypoxic signaling pathway in hepatoma cells, which may contribute to its anticancer effect. Thus, disulfiram could be used to treat solid tumors that grow in a HIF-dependent manner.

Mad1 mediates hypoxia-induced doxorubicin resistance in colon cancer cells by inhibiting mitochondrial function.

Cancer cells acquire resistance to chemotherapy under hypoxia, which is mainly driven by the transcription factor HIF (hypoxia-inducible factor). Yet, it is uncertain which molecules mediate such resistance. While profiling gene expression in colon cancer cells, we found that Mad1 (MAX dimerization protein 1) is substantially induced during hypoxia. The hypoxic induction of Mad1 was confirmed by RT-PCR and Western blotting. The Mad1 expression was attenuated by HIF-1α small interfering (si) RNAs, but less so by HIF-2α siRNAs. Moreover, luciferase reporter and chromatin immunoprecipitation analyses revealed that HIF-1 transactivates the MAD1 gene by directly targeting a putative hypoxia-response element in the MAD1 promoter. We next investigated if Mad1 is responsible for the hypoxia-induced drug resistance. We treated colon cancer cells with doxorubicin and found that the cells under hypoxia survived more than those under normoxia. The doxorubicin resistance was not induced in Mad1-knocked-down cells even under hypoxia. Mad1 knockdown reactivated the caspase-9/caspase-3/PARP apoptotic pathway under hypoxia. Moreover, doxorubicin-induced production of reactive oxygen species was significantly reduced under hypoxia, which was reversed by Mad1 knockdown. During hypoxia, mitochondria became bigger in size and less active in respiration, both of which were attenuated by Mad1 knockdown. These data indicate that hypoxia-induced Mad1 lowers doxorubicin-stimulated generation of reactive oxygen species through mitochondrial inhibition and subsequently contributes to tumor resistance to doxorubicin. Therefore, Mad1 could be a potential target for sensitizing cancer cells to redox-cycling drugs such as doxorubicin.

Hypoxia-inducible factor 1 mediates nasal polypogenesis by inducing epithelial-to-mesenchymal transition.

RATIONALE: Nasal polyposis implies a refractory clinical course in case of chronic rhinosinusitis (CRS). Although hypoxia is believed to be associated with nasal polyposis, little is known about the mechanism underlying polypogenesis. OBJECTIVES: To determine if hypoxia drives nasal polyposis by epithelial-to-mesenchymal transition (EMT). METHODS: Immunoblotting, immunofluorescence, flow cytometry, and real-time polymerase chain reaction were performed to evaluate EMT and hypoxic markers in human nasal epithelial cells (hNECs) and in sinonasal tissues from patients with CRS with or without polyps. In addition, the effects of hypoxia-inducible factor (HIF)-1α inhibitors on nasal polypogenesis were investigated in a murine model. MEASUREMENTS AND MAIN RESULTS: E-cadherin and α-smooth muscle actin (α-SMA) were down-regulated and up-regulated, respectively, in patients with polyps as compared with patients without polyps. Under hypoxia, hNECs transformed to a mesenchymal shape, and demonstrated representative changes in EMT markers; that is, mesenchymal markers (α-SMA, vimentin, and twist) increased but epithelial markers (E-cadherin and ß-catenin) decreased. Mechanistically, E-cadherin level was recovered in hypoxia by silencing HIF-1α and decreased in normoxia by expressing HIF-1α. Furthermore, hypoxia was found to down-regulate PP2Ac phosphatase and up-regulate pSmad3, which led to α-SMA induction. In CRS sinonasal specimens, HIF-1α expression was found to correlate with E-cadherin loss and α-SMA expression. Finally, HIF-1α inhibitors suppressed nasal polypogenesis in a murine model. CONCLUSIONS: hNECs undergo EMT during hypoxia and this process is critically mediated by HIF-1α and pSmad3. This study shows that hypoxia-induced EMT is likely to contribute to nasal polyposis in CRS, and suggests that HIF-1α be viewed as a therapeutic target for nasal polyposis.