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PDF - 587K, Supplementary Materials and Methods; Supplementary Figure Legend. Supplementary Fig. 1. The effect of over-expressing and/or ligand activation of PPAR/ on cell cycle progression in MDA-MB-231 and MCF7 cells. Supplementary Fig. 2. The effect of over-expressing PPARbeta/? and/or ligand activation of PPARbeta/? on staurosporine induced PARP cleavage in MDA-MB-231 and MCF7 cells. Supplementary Fig. 3. The effect of over-expressing PPARbeta/? and/or ligand activation of PPARbeta/? on UVB-induced PARP cleavage in MDA-MB-231 and MCF7 cells. Supplementary Fig. 4. The effect of over-expressing PPARbeta/? and/or ligand activation of PPARbeta/? on xenograft histopathology and necrosis.

The effect of activation and overexpression of the nuclear receptor PPAR-β/δ in human MDA-MB-231 (estrogen receptor–negative; ER−) and MCF7 (estrogen-receptor-positive; ER+) breast cancer cell lines was examined. Target gene induction by ligand activation of PPAR-β/δ was increased by overexpression of PPAR-β/δ compared with controls. Overexpression of PPAR-β/δ caused a decrease in cell proliferation in MCF7 and MDA-MB-231 cells compared with controls, whereas ligand activation of PPAR-β/δ further inhibited proliferation of MCF7 but not MDA-MB-231 cells. Overexpression and/or ligand activation of PPAR-β/δ in MDA-MB-231 or MCF7 cells had no effect on experimental apoptosis. Decreased clonogenicity was observed in both MDA-MB-231 and MCF7 overexpressing PPAR-β/δ in response to ligand activation of PPAR-β/δ as compared with controls. Ectopic xenografts developed from MDA-MB-231 and MCF7 cells overexpressing PPAR-β/δ were significantly smaller, and ligand activation of PPAR-β/δ caused an even greater reduction in tumor volume as compared with controls. Interestingly, the decrease in MDA-MB-231 tumor size after overexpressing PPAR-β/δ and ligand activation of PPAR-β/δ correlated with increased necrosis. These data show that ligand activation and/or overexpression of PPAR-β/δ in two human breast cancer cell lines inhibits relative breast cancer tumorigenicity and provide further support for the development of ligands for PPAR-β/δ to specifically inhibit breast carcinogenesis. These new cell-based models will be invaluable tools for delineating the role of PPAR-β/δ in breast cancer and evaluating the effects of PPAR-β/δ agonists. Mol Cancer Ther; 13(4); 1008–17. ©2014 AACR.

Excessive, chronic alcohol consumption can lead to alcoholic liver disease. The etiology of alcoholic liver disease is multifactorial and is influenced by alterations in gene expression and changes in fatty acid metabolism, oxidative stress, and insulin resistance. These events can lead to steatosis, fibrosis, and eventually to cirrhosis and liver cancer. Many of these functions are regulated by peroxisome proliferator-activated receptors (PPARs). Thus, it is not surprising that PPARs can modulate the mechanisms that cause alcoholic liver disease. While the roles of PPARα and PPARγ are clearer, the role of PPARβ/δ in alcoholic liver disease requires further clarification. This review summarizes the current understanding based on recent studies that indicate that PPARβ/δ can likely be targeted for the treatment and/or the prevention of alcoholic liver disease.

High-resolution mass spectrometry (MS) has advanced the study of metabolism in living systems by allowing many metabolites to be measured in a single experiment. Although improvements in mass detector sensitivity have facilitated the detection of greater numbers of analytes, compound identification strategies, feature reduction software, and data sharing have not kept up with the influx of MS data. Here, we discuss the ongoing challenges with MS-based metabolomics, including de novo metabolite identification from mass spectra, differentiation of metabolites from environmental contamination, chromatographic separation of isomers, and incomplete MS databases. Because of their popularity and sensitive detection of small molecules, this review focuses on the challenges of liquid chromatography-mass spectrometry–based methods. We then highlight important instrumentational, experimental, and computational tools that have been created to address these challenges and how they have enabled the advancement of metabolomics research.

Cannabidiolic acid (CBDA) can activate peroxisome proliferator-activated receptor-α (PPARα) and PPARγ. Whether CBDA can activate PPARβ/δ has not been examined sufficiently to date. Since previous studies showed that triple-negative breast cancer cells respond to activation of PPARβ/δ, the present study examined the effect of CBDA in MDA-MB-231 cells and compared the activities of CBDA with known PPARβ/δ agonists/antagonists. Expression of the PPARβ/δ target genes angiopoietin-like 4 (ANGPTL4) and adipocyte differentiation-related protein (ADRP) was increased by CBDA. Interestingly, ligand activation of PPARβ/δ with GW501516 caused an increase in expression of both ANGPTL4 and ADRP, but the magnitude of this effect was markedly increased when co-treated with CBDA. Specificity of these effects were confirmed by showing that CBDA-induced expression of ANGPTL4 and ADRP is mitigated in the presence of either a PPARβ/δ antagonist or an inverse agonist. Results from these studies suggest that CBDA can synergize with PPARβ/δ and might interact with endogenous agonists that modulate PPARβ/δ function.

Perfluorooctane sulfonate (PFOS) is a stable environmental contaminant that can activate peroxisome proliferator-activated receptor alpha (PPARα). In the present work, the specific role of mouse and human PPARα in mediating the hepatic effects of PFOS was examined in short-term studies using wild type, Ppara-null and PPARA-humanized mice. Mice fed 0.006 % PFOS for seven days (~10 mg/kg/day), or 0.003 % PFOS for twenty-eight days (~5 mg/kg/day), exhibited higher liver and serum PFOS concentrations compared to controls. Relative liver weights were also higher following exposure to dietary PFOS in all three genotypes as compared vehicle fed control groups. Histopathological examination of liver sections from mice treated for twenty-eight days with 0.003 % PFOS revealed a phenotype consistent with peroxisome proliferation, in wild-type and PPARA-humanized mice that was not observed in Ppara-null mice. With both exposures, expression of the PPARα target genes, Acox1, Cyp4a10, was significantly increased in wild type mice but not in Ppara-null or PPARA-humanized mice. By contrast, expression of the constitutive androstane receptor (CAR) target gene, Cyp2b10, and the pregnane × receptor (PXR) target gene, Cyp3a11, were higher in response to PFOS administration in all three genotypes compared to controls for both exposure periods. These results indicate that mouse PPARα can be activated in the liver by PFOS causing increased expression of Acox1, Cyp4a10 and histopathological changes in the liver. While histopathological analyses indicated the presence of mouse PPARα-dependent hepatic peroxi-some proliferation in wild-type (a response associated with activation of PPARα) and a similar phenotype in PPARA-humanized mice, the lack of increased Acox1 and Cyp4a10 mRNA by PFOS in PPARA-humanized mice indicates that the human PPARα was not as responsive to PFOS as mouse PPARα with this dose regimen. Moreover, results indicate that hepatomegaly caused by PFOS does not require mouse or human PPARα and could be due to effects induced by activation of CAR and/or PXR.

The peroxisome proliferator-activated-β/δ (PPARβ/δ) was identified in 1994, but not until 1999 was PPARβ/δ suggested to be involved in carcinogenesis. Initially, it was hypothesized that expression of PPARβ/δ was increased during colon cancer progression, which led to increased transcription of yet-to-be confirmed target genes that promote cell proliferation and tumorigenesis. It was also hypothesized at this time that lipid-metabolizing enzymes generated lipid metabolites that served as ligands for PPARβ/δ. These hypothetical mechanisms were attractive because they potentially explained how non-steroidal anti-inflammatory drugs inhibited tumorigenesis by potentially limiting the concentration of endogenous PPARβ/δ ligands that could activate this receptor that was increased in cancer cells. However, during the last 20 years, considerable research was undertaken describing expression of PPARβ/δ in normal and cancer cells that has led to a significant impact on the mechanisms by which PPARβ/δ functions in carcinogenesis. Whereas results from earlier studies led to much uncertainty about the role of PPARβ/δ in cancer, more recent analyses of large databases have revealed a more consistent understanding. The focus of this review is on the fundamental level of PPARβ/δ expression in normal tissues and cancerous tissue as described by studies during the past two decades and what has been delineated during this timeframe about how PPARβ/δ expression influences carcinogenesis, with an emphasis on colon cancer.

The human microbiome is composed of four major areas including intestinal, skin, vaginal, and oral microbiomes, with each area containing unique species and unique functionalities. The human microbiome may be modulated with prebiotics, probiotics, and postbiotics to potentially aid in the treatment of diseases like irritable bowel syndrome, bacterial vaginosis, atopic dermatitis, gingivitis, obesity, or cancer. There is also potential for many of the inhabitants of the human microbiome to directly modulate host gene expression and modulate host detoxifying enzyme activity like cytochrome P450s (CYPs), dehydrogenases, and carboxylesterases. Therefore, the microbiome may be important to consider during drug discovery, risk assessment, and dosing regimens for various diseases given that the human microbiome has been shown to impact host detoxification processes.

Considerable progress has been made during the past 20 years towards elucidating the role of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in skin cancer. In 1999, the original notion that PPARβ/δ was involved with epithelial cell function was postulated based on a correlation between PPARβ/δ expression and the induction of messenger RNAs encoding proteins that mediate terminal differentiation in keratinocytes. Subsequent studies definitively revealed that PPARβ/δ could induce terminal differentiation and inhibit proliferation of keratinocytes. Molecular mechanisms have since been discovered to explain how this nuclear receptor can be targeted for preventing and treating skin cancer. This includes the regulation of terminal differentiation, mitotic signaling, endoplasmic reticulum stress, and cellular senescence. Interestingly, the effects of activating PPARβ/δ can preferentially target keratinocytes with genetic mutations associated with skin cancer. This review provides the history and current understanding of how PPARβ/δ can be targeted for both nonmelanoma skin cancer and melanoma and postulates how future approaches that modulate PPARβ/δ signaling may be developed for the prevention and treatment of these diseases.

Ppara-null and PPARA-humanized mice are refractory to hepatocarcinogenesis caused by the peroxisome proliferator-activated receptor-α (PPARα) agonist Wy-14,643. However, the duration of these earlier studies was limited to approximately 1 year of treatment, and the ligand used has a higher affinity for the mouse PPARα compared to the human PPARα. Thus, the present study examined the effect of long-term administration of a potent, high-affinity human PPARα agonist (GW7647) on hepatocarcinogenesis in wild-type, Ppara-null, or PPARA-humanized mice. In wild-type mice, GW7647 caused hepatic expression of known PPARα target genes, hepatomegaly, hepatic MYC expression, hepatic cytotoxicity, and a high incidence of hepatocarcinogenesis. By contrast, these effects were essentially absent in Ppara-null mice or diminished in PPARA-humanized mice, although hepatocarcinogenesis was observed in both genotypes. Enhanced fatty change (steatosis) was also observed in both Ppara-null and PPARA-humanized mice independent of GW7647. PPARA-humanized mice administered GW7647 also exhibited increased necrosis after 5 weeks of treatment. Results from these studies demonstrate that the mouse PPARα is required for hepatocarcinogenesis induced by GW7647 administered throughout adulthood. Results also indicate that a species difference exists between rodents and human PPARα in the response to ligand activation of PPARα. The hepatocarcinogenesis observed in control and treated Ppara-null mice is likely mediated in part by increased hepatic fatty change, whereas the hepatocarcinogenesis observed in PPARA-humanized mice may also be due to enhanced fatty change and cytotoxicity that could be influenced by the minimal activity of the human PPARα in this mouse line on downstream mouse PPARα target genes. The Ppara-null and PPARA-humanized mouse models are valuable tools for examining the mechanisms of PPARα-induced hepatocarcinogenesis, but the background level of liver cancer must be controlled for in the design and interpretation of studies that use these mice.

Evidence suggests that species differences exist between rodents and humans in their biological responses to ligand activation of PPARα. Moreover, neonatal/postnatal rodents may be more sensitive to the effects of activating PPARα. Thus, the present studies examined the effects of chronic ligand activation of PPARα initiated during early neonatal development and continued into adulthood on hepatocarcinogenesis in mice. Wild-type, Ppara-null, or PPARA-humanized mice were administered a potent, high-affinity human PPARα agonist GW7647, and cohorts of mice were examined over time. Activation of PPARα with GW7647 increased expression of known PPARα target genes in liver and was associated with hepatomegaly, increased hepatic cytotoxicity and necrosis, increased expression of hepatic MYC, and a high incidence of hepatocarcinogenesis in wild-type mice. These effects did not occur or were largely diminished in Ppara-null and PPARA-humanized mice, although background levels of hepatocarcinogenesis were also noted in both Ppara-null and PPARA-humanized mice. More fatty change (steatosis) was also observed in both Ppara-null and PPARA-humanized mice independent of GW7647 administration. Results from these studies indicate that the mouse PPARα is required to mediate hepatocarcinogenesis induced by GW7647 in mice and that activation of the human PPARα with GW7647 in PPARA-humanized mice are diminished compared with wild-type mice. Ppara-null and PPARA-humanized mice are valuable tools for examining species differences in the mechanisms of PPARα-induced hepatocarcinogenesis, but background levels of liver cancer observed in aged Ppara-null and PPARA-humanized mice must be considered when interpreting results from studies that use these models. These results also demonstrate that early life exposure to a potent human PPARα agonist does not enhance sensitivity to hepatocarcinogenesis.

Perfluorooctane sulfonate (PFOS) is a persistent environmental chemical whose biological effects are mediated by multiple mechanisms. Recent evidence suggests that the gut microbiome may be directly impacted by and/or alter the fate and effects of environmental chemicals in the host. Thus, the aim of this study was to determine whether PFOS influences the gut microbiome and its metabolism, and the host metabolome. Four groups of male C57BL/6 J mice were fed a diet with or without 0.003 %, 0.006 %, or 0.012 % PFOS, respectively. 16S rRNA gene sequencing, metabolomic, and molecular analyses were used to examine the gut microbiota of mice after dietary PFOS exposure. Dietary PFOS exposure caused a marked change in the gut microbiome compared to controls. Dietary PFOS also caused dose-dependent changes in hepatic metabolic pathways including those involved in lipid metabolism, oxidative stress, inflammation, TCA cycle, glucose, and amino acid metabolism. Changes in the metabolome correlated with changes in genes that regulate these pathways. Integrative analyses also demonstrated a strong correlation between the alterations in microbiota composition and host metabolic profiles induced by PFOS. Further, using isolated mouse cecal contents, PFOS exposure directly affected the gut microbiota metabolism. Results from these studies demonstrate that the molecular and biochemical changes induced by PFOS are mediated in part by the gut microbiome, which alters gene expression and the host metabolome in mice.

The tumor microenvironment can be hypoxic, acidic, and deficient in nutrients. This can cause the metabolism of tumor cells as well as the neighboring stromal cells to be remodelled to facilitate tumor survival, proliferation, and metastasis. Abnormal tumor lipid metabolism is a fairly new field, which has received attention in the past few years. Cross-talk between tumor cells and tumor associated stromal cells modulates the high metabolic needs of the tumor. Fatty acid turnover is high in tumor cells to meet the energy as well as synthetic requirements of the growing tumor. Lipolysis of lipids stored in lipid droplets was earlier considered to be solely carried out by cytosolic lipases. However recent studies demonstrate that lipophagy (autophagic degradation of lipids by acidic lipases) serves as an alternate pathway for the degradation of lipid droplets. Involvement of lipophagy in lipid turnover makes it a crucial player in tumorigenesis and metastasis. In this review we discuss the metabolic reprogramming of tumor cells with special focus on lipid metabolism. We also address the lipid turnover machinery in the tumor cell, especially the lipophagic pathway. Finally, we integrate the current understanding of lipophagy with tumor lipid metabolism.

To examine the functional role of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) and PPARγ in skin cancer, stable cell lines were created in the A431 human squamous cell carcinoma cell line. Expression of PPAR target genes was greatly enhanced in response to ligand activation of PPARβ/δ or PPARγ in A431 cells expressing these receptors. PPARβ/δ expression blocked the cell cycle at the G2/M phase, and this effect was increased by ligand activation. Ligand activation of PPARβ/δ markedly inhibited clonogenicity as compared to vehicle-treated controls. Similarly, ligand activation of PPARγ in A431 cells expressing PPARγ resulted in reduced clonogenicity. Expression of either PPARβ/δ or PPARγ markedly reduced tumor volume in ectopic xenografts, while ligand activation of these receptors had little further influence on tumor volume. Collectively, these studies demonstrate that stable expression and activation of PPARβ/δ or PPARγ in A431 cells led to reduced tumorigenicity. Importantly, PPAR expression or ligand activation had major impacts on clonogenicity and/or tumor volume. Thus, PPARβ/δ or PPARγ could be therapeutically targeted for the treatment of squamous cell carcinomas.

A number of industrial chemicals and therapeutic agents cause liver tumors in rats and mice by activating the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). The molecular and cellular events by which PPARα activators induce rodent hepatocarcinogenesis have been extensively studied elucidating a number of consistent mechanistic changes linked to the increased incidence of liver neoplasms. The weight of evidence relevant to the hypothesized mode of action (MOA) for PPARα activator-induced rodent hepatocarcinogenesis is summarized here. Chemical-specific and mechanistic data support concordance of temporal and dose–response relationships for the key events associated with many PPARα activators. The key events (KE) identified in the MOA are PPARα activation (KE1), alteration in cell growth pathways (KE2), perturbation of hepatocyte growth and survival (KE3), and selective clonal expansion of preneoplastic foci cells (KE4), which leads to the apical event–increases in hepatocellular adenomas and carcinomas (KE5). In addition, a number of concurrent molecular and cellular events have been classified as modulating factors, because they potentially alter the ability of PPARα activators to increase rodent liver cancer while not being key events themselves. These modulating factors include increases in oxidative stress and activation of NF-kB. PPARα activators are unlikely to induce liver tumors in humans due to biological differences in the response of KEs downstream of PPARα activation. This conclusion is based on minimal or no effects observed on cell growth pathways and hepatocellular proliferation in human primary hepatocytes and absence of alteration in growth pathways, hepatocyte proliferation, and tumors in the livers of species (hamsters, guinea pigs and cynomolgus monkeys) that are more appropriate human surrogates than mice and rats at overlapping dose levels. Despite this overwhelming body of evidence and almost universal acceptance of the PPARα MOA and lack of human relevance, several reviews have selectively focused on specific studies that, as discussed, contradict the consensus opinion and suggest uncertainty. In the present review, we systematically address these most germane suggested weaknesses of the PPARα MOA.

Choline is an essential nutrient utilized for phosphatidylcholine biosynthesis and lipoprotein packaging and secretion. Recently, choline supplementation has been used by athletes and the public for weight loss. However, the potential toxicological impact of choline dietary supplementation requires further investigation. This study examined the effects of choline dietary supplementation in Sprague Dawley rats for 4 weeks. Rats were fed diets containing basal choline levels (control) or 5-, 10-, or 15-fold (5×, 10×, or 15×) basal diet concentration. In groups fed choline-supplemented diets, there were no toxicologically relevant findings in clinical observations, food intake, clinical chemistry, liver weights, or liver histopathology. However, decreased mean body weights (8.5–10.2%) and body weight gains (24–31%) were noted for the 10× choline-supplemented (females only) and 15× choline-supplemented (both sexes) groups relative to the control groups from day 3 onward. These body weight effects were not related to a persistent reduction in average food intake. Serum cholesterol was increased in the 15× choline-supplemented male rats relative to the controls, an expected effect of choline supplementation; however, there were no changes in the serum cholesterol of female rats. Serum choline concentrations were increased in female rats relative to the male rats across all treatment groups. The maximum tolerated dose for male and female rats were the 15× and 10× choline supplements, respectively, based on decreased mean body weight and body weight gains. This study supported the conclusions of a clinical trial that showed a high choline diet can decrease body weight in humans.

Skin tumorigenesis results from DNA damage, increased inflammation, and evasion of apoptosis. The peroxisome proliferator-activated receptors (PPARs) can modulate these mechanisms in non-melanoma skin cancer. However, limited data exists regarding the role of PPARs in melanoma. This study examined the effect of proliferator-activated receptor-β/δ (PPARβ/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARβ/δ or PPARγ enhanced ligand-induced expression of a PPARβ/δ/PPARγ target gene in UACC903 cell lines as compared with controls. The induction of target gene expression by ligand activation of PPARγ was not altered by overexpression of PPARβ/δ, or vice versa. Stable overexpression of either PPARβ/δ or PPARγ reduced the percentage of cells in the G1 and S phase of the cell cycle, and increased the percentage of cells in the G2/M phase of the cell cycle in UACC903 cell lines as compared with controls. Ligand activation of PPARβ/δ did not further alter the distribution of cells within each phase of the cell cycle. By contrast, ligand activation of PPARγ enhanced these changes in stable UACC903 cells overexpressing PPARγ compared with controls. Stable overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited ectopic xenograft tumorigenicity derived from UACC903 melanoma cells as compared with controls, and this was likely due in part to induction of apoptosis. Results from these studies demonstrate the antitumorigenic effects of both PPARβ/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

The peroxisome proliferator‐activated receptor‐β/δ (PPAR β/δ) is known to have multiple anti‐inflammatory effects, typically observed in endothelial cells, macrophages, T cells and B cells. Despite the fact that mast cells are important mediators of inflammation, to date, the role of PPAR β/δ in mast cells has not been examined. Hence, the present study examined the hypothesis that PPAR β/δ modulates mast cell phenotype. Bone‐marrow‐derived mast cells (BMMCs) and peritoneal mast cells from Pparβ/δ +/+ mice expressed higher levels of high‐affinity IgE receptor (Fcε RI) compared with Pparβ/δ −/− mice. BMMCs from Pparβ/δ +/+ mice also exhibited dense granules, associated with higher expression of enzymes and proteases compared with Pparβ/δ −/− mice. Resting BMMCs from Pparβ/δ +/+ mice secreted lower levels of inflammatory cytokines, associated with the altered activation of phospholipase Cγ1 and extracellular signal‐regulated kinases compared with Pparβ/δ −/− mice. Moreover, the production of cytokines by mast cells induced by various stimuli was highly dependent on PPAR β/δ expression. This study demonstrates that PPAR β/δ is an important regulator of mast cell phenotype.

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients.

Alcoholic liver disease is a pathological condition caused by overconsumption of alcohol. Because of the high morbidity and mortality associated with this disease, there remains a need to elucidate the molecular mechanisms underlying its etiology and to develop new treatments. Because peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) modulates ethanol-induced hepatic effects, the present study examined alterations in gene expression that may contribute to this disease. Chronic ethanol treatment causes increased hepatic CYP2B10 expression inPparβ/δ+/+ mice but not in Pparβ/δ−/− mice. Nuclear and cytosolic localization of the constitutive androstane receptor (CAR), a transcription factor known to regulate Cyp2b10 expression, was not different between genotypes. PPARγ co-activator 1α, a co-activator of both CAR and PPARβ/δ, was up-regulated in Pparβ/δ+/+ liver following ethanol exposure, but not in Pparβ/δ−/− liver. Functional mapping of the Cyp2b10 promoter and ChIP assays revealed that PPARβ/δ-dependent modulation of SP1 promoter occupancy up-regulated Cyp2b10 expression in response to ethanol. These results suggest that PPARβ/δ regulates Cyp2b10 expression indirectly by modulating SP1 and PPARγ co-activator 1α expression and/or activity independent of CAR activity. Ligand activation of PPARβ/δ attenuates ethanol-induced Cyp2b10 expression in Pparβ/δ+/+ liver but not in Pparβ/δ−/− liver. Strikingly, Cyp2b10 suppression by ligand activation of PPARβ/δ following ethanol treatment occurred in hepatocytes and was mediated by paracrine signaling from Kupffer cells. Combined, results from the present study demonstrate a novel regulatory role of PPARβ/δ in modulating CYP2B10 that may contribute to the etiology of alcoholic liver disease.

The drug metabolism field has long recognized the beneficial and sometimes deleterious influence of microbiota in the absorption, distribution, metabolism, and excretion of drugs. Early pioneering work with the sulfanilamide precursor prontosil pointed toward the necessity not only to better understand the metabolic capabilities of the microbiota but also, importantly, to identify the specific microbiota involved in the generation and metabolism of drugs. However, technological limitations important for cataloging the microbiota community as well as for understanding and/or predicting their metabolic capabilities hindered progress. Current advances including mass spectrometry-based metabolite profiling as well as culture-independent sequence-based identification and functional analysis of microbiota have begun to shed light on microbial metabolism. In this review, case studies will be presented to highlight key aspects (e.g., microbiota identification, metabolic function and prediction, metabolite identification, and profiling) that have helped to clarify how the microbiota might impact or be impacted by drug metabolism. Lastly, a perspective of the future of this field is presented that takes into account what important knowledge is lacking and how to tackle these problems.

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) is a nuclear receptor that regulates differentiation, inflammation, lipid metabolism, extracellular matrix remodeling, and angiogenesis in multiple tissues. These pathways are also central to the pathogenesis of age-related macular degeneration (AMD), the leading cause of vision loss globally. With the goal of identifying signaling pathways that may be important in the development of AMD, we investigated the impact of PPARβ/δ activation on ocular tissues affected in the disease. PPARβ/δ is expressed and can be activated in AMD vulnerable cells, including retinal pigment epithelial (RPE) and choroidal endothelial cells. Further, PPARβ/δ knockdown modulates AMD-related pathways selectively. Specifically, genetic ablation of Pparβ/δ in aged mice resulted in exacerbation of several phenotypic features of early dry AMD, but attenuation of experimentally induced choroidal neovascular (CNV) lesions. Antagonizing PPARβ/δ in both in vitro angiogenesis assays and in the in vivo experimentally induced CNV model, inhibited angiogenesis and angiogenic pathways, while ligand activation of PPARβ/δ, in vitro, decreased RPE lipid accumulation, characteristic of dry AMD. This study demonstrates for the first time, selective regulation of a nuclear receptor in the eye and establishes that selective targeting of PPARβ/δ may be a suitable strategy for treatment of different clinical sub-types of AMD.

An explosion of knowledge on the molecular and cellular mechanisms that mediate carcinogenesis has occurred in recent years. Although cancer has existed for over a million years in the human species, effective cures for most cancers that target molecular and cellular pathways have not been achieved. Multiple cellular targets have been examined for preventing or treating cancers including, but not limited to, transcription factors, kinase-mediated cell signaling pathways, and more recently epigenetic targeting of oncogenes and tumor suppressors, and immunomodulation such as chimeric antigen receptor-T cells. Even as the state of knowledge of cancer mechanisms increases, there is considerable room for improvement in preventing and treating cancers. Understanding how a normal cell is transformed into a cancer cell is known but there is considerable tissue and cell type specificity. This has given rise to the field of precision medicine as applied to cancer therapy. Thus, while the development of preventive and treatment regimens has increased, there are certain obstacles that need to be overcome in order to decrease cancer incidence and increase survival of cancer patients. The purpose of this review is to summarize the advances made in cancer biology and how these advances have been used to develop, and hinder, preventive, and therapeutic strategies for cancer.

A common theme in chemically induced mammalian toxicity is the ability of certain chemicals to mediate their effects through activation of members of the nuclear receptor superfamily or the aryl hydrocarbon receptor (AHR), a basic helix–loop–helix/PAS ligand-activated transcription factor. This in turn leads to a myriad of changes in gene expression as these receptors bind to their cognate response elements. Receptor-induced toxicity can be divided into four basic mechanisms, the first being the disruption of the activation pathways involved in normal development and physiological processes. The second mechanism of receptor-mediated toxicity is the ability of the xenobiotic-induced receptor to induce a spectrum of transcriptional activity not normally observed. The third general mechanism of toxicity involves the ability of an activated receptor to increase levels of enzymes that metabolize the receptor ligand to toxic metabolites. The fourth more recently studied mechanism of toxicity involves the interaction of the gut microbiota with host receptors to either modify or directly mediate the toxicity response. The mechanisms of PPARα/β-, estrogen receptor-, and the AHR-mediated toxicity are described in detail to illustrate the various mechanisms that have been experimentally determined.

Ppard(-/-) mice exhibit smaller litter size compared with Ppard(+/+) mice. To determine whether peroxisome proliferator-activated receptor-D (PPARD) could possibly influence this phenotype, the role of PPARD in testicular biology was examined. Atrophic testes and testicular degeneration were observed in Ppard(-/-) mice compared with Ppard(+/+) mice, indicating that PPARD modulates spermatogenesis. Higher expression of p27 and decreased expression of proliferating cellular nuclear antigen in Sertoli cells were observed in Ppard(+/+) mice as compared with Ppard(-/-) mice, and these were associated with decreased Sertoli cell number in Ppard(+/+) mice. Cyclin D1 and cyclin D2 expression was lower in Ppard(+/+) as compared with Ppard(-/-) mice. Ligand activation of PPARD inhibited proliferation of a mouse Sertoli cell line, TM4, and an inverse agonist of PPARD (DG172) rescued this effect. Temporal inhibition of extracellular signal-regulated kinase (ERK) activation by PPARD in the testis was observed in Ppard(+/+) mice and was associated with decreased serum follicle-stimulating hormone and higher claudin-11 expression along the blood-testis barrier. PPARD-dependent ERK activation also altered expression of claudin-11, p27, cyclin D1, and cyclin D2 in TM4 cells, causing inhibition of cell proliferation, maturation, and formation of tight junctions in Sertoli cells, thus confirming a requirement for PPARD in accurate Sertoli cell function. Combined, these results reveal for the first time that PPARD regulates spermatogenesis by modulating the function of Sertoli cells during early testis development.

The role of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in cancer remains contentious due in large part to divergent publications indicating opposing effects in different rodent and human cell culture models. During the past 10 years, some facts regarding PPARβ/δ in cancer have become clearer, while others remain uncertain. For example, it is now well accepted that (1) expression of PPARβ/δ is relatively lower in most human tumors as compared to the corresponding non-transformed tissue, (2) PPARβ/δ promotes terminal differentiation, and (3) PPARβ/δ inhibits pro-inflammatory signaling in multiple in vivo models. However, whether PPARβ/δ is suitable to target with natural and/or synthetic agonists or antagonists for cancer chemoprevention is hindered because of the uncertainty in the mechanism of action and role in carcinogenesis. Recent findings that shed new insight into the possibility of targeting this nuclear receptor to improve human health will be discussed.

We investigate crosstalk between cancer cells and stromal myeloid cells. We find that Lewis lung carcinoma cells significantly induce PPAR beta/delta activity in myeloid cells in vitro and in vivo. Myeloid cell-specific knockout of PPAR beta/delta results in impaired growth of implanted tumors, and this is restored by adoptive transfer of wild-type myeloid cells. We find that IL-10 is a downstream effector of PPAR beta/delta and facilitates tumor cell invasion and angiogenesis. This observation is supported by the finding that the CD11b(low)IL-10(+) pro-tumoral myeloid cell is scarcely detected in tumors from myeloid-cell-specific PPAR beta/delta knockout mice, where vessel densities are also decreased. Fatty acid synthase (FASN) is shown to be an upstream regulator of PPAR beta/delta in myeloid cells and is induced by M-CSF secreted from tumor cells. Our study gives insight into how cancer cells influence myeloid stromal cells to get a pro-tumoral phenotype.

Perfluorooctane sulfonate (PFOS) is an environmentally persistent chemical. Dietary 100 ppm PFOS fed to male mice and rats for 4 weeks caused hepatic steatosis through an unknown mechanism. Choline deficient diets can cause hepatic steatosis. A hepatic choline:PFOS ion complex was hypothesized to cause this effect in mice. This study tested whether dietary choline supplementation attenuates PFOS-induced hepatic steatosis in rats. Sprague Dawley rats (12/sex/group) were fed control, choline supplemented (CS), 100 ppm PFOS, or 100 ppm PFOS + CS diets for 3 weeks. Male rats fed both PFOS-containing diets had decreased serum cholesterol and triglycerides (TGs) on days 9, 16, and/or 23 and increased hepatic free fatty acids and TG (ie, steatosis). Female rats fed both PFOS diets had decreased serum cholesterol on days 9 and 16 and decreased hepatic free fatty acid and TG at termination (ie, no steatosis). Liver PFOS concentrations were similar for both sexes. Liver choline concentrations were increased in male rats fed PFOS (±CS), but the increase was lower in the PFOS + CS group. Female liver choline concentrations were not altered by any diet. These findings demonstrate a clear sex-related difference in PFOS-induced hepatic steatosis in the rat. Additional evaluated mechanisms (ie, nuclear receptor activation, mRNA upregulation, and choline kinase activity inhibition) did not appear to be involved in the hepatic steatosis. Dietary PFOS (100 ppm) induced hepatic steatosis in male, but not female, rats that was not attenuated by choline supplementation. The mechanism of lipid accumulation and the sex-related differences warrant further investigation.

Energy homeostasis and oncogenic signaling are critical determinants of the growth of human liver cancer cells, providing a strong rationale to elucidate the regulatory mechanisms for these systems. A new study reports that loss of solute carrier family 13 member 5, which transports citrate across cell membranes, halts liver cancer cell growth by altering both energy production and mammalian target of rapamycin signaling in human liver cancer cell lines and in both an in vitro and in vivo model of liver tumors, suggesting a new target for liver cancer chemoprevention and/or chemotherapy.

Whether peroxisome proliferator-activated receptor β/δ (PPARβ/δ) reduces skin tumorigenesis by altering aryl hydrocarbon receptor (AHR)-dependent activities was examined. Polycyclic aromatic hydrocarbons (PAH) increased expression of cytochrome P4501A1 (CYP1A1), CYP1B1 and phase II xenobiotic metabolizing enzymes in wild-type skin and keratinocytes. Surprisingly, this effect was not found in Pparβ/δ-null skin and keratinocytes. Pparβ/δ-null keratinocytes exhibited decreased AHR occupancy and histone acetylation on the Cyp1a1 promoter in response to a PAH compared with wild-type keratinocytes. Bisulfite sequencing of the Cyp1a1 promoter and studies using a DNA methylation inhibitor suggest that PPARβ/δ promotes demethylation of the Cyp1a1 promoter. Experiments with human HaCaT keratinocytes stably expressing shRNA against PPARβ/δ also support this conclusion. Consistent with the lower AHR-dependent activities in Pparβ/δ-null mice compared with wild-type mice, 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis was inhibited in Pparβ/δ-null mice compared with wild-type. Results from these studies demonstrate that PPARβ/δ is required to mediate complete carcinogenesis by DMBA. The mechanisms underlying this PPARβ/δ-dependent reduction of AHR signaling by PAH are not due to alterations in the expression of AHR auxiliary proteins, ligand binding or AHR nuclear translocation between genotypes, but are likely influenced by PPARβ/δ-dependent demethylation of AHR target gene promoters including Cyp1a1 that reduces AHR accessibility as shown by reduced promoter occupancy. This PPARβ/δ/AHR crosstalk is unique to keratinocytes and conserved between mice and humans.

Background Five-year survival after the surgical treatment of esophageal cancer has traditionally been reported to be as low as 15%. More recently, the improvement of clinical staging involving PET/CT and the introduction of neoadjuvant chemo-radiation have each altered the survival outcomes of patients with this lethal disease. The impact of these factors on survival trends has not been well described in literature. The aim of this study was to analyze the survival trends after esophagectomy for esophageal adenocarcinoma at a high-volume center. Methods The study population consisted of 471 consecutive patients undergoing esophagectomy for esophageal adenocarcinoma at a university-based medical center between January 1, 2000 and July 31, 2017. Clinical variables were collected for three groups based on the date of esophagectomy and were compared (Group 1: 2000–2004, Group 2: 2005–2011, Group 3: 2012–2017). Survival was compared via the Kaplan-Meier (KM) method. Results The 471 patients had a median age of 64.0 years (range 27.0–86.2) and 395/471 (84%) were male. Dysphagia (282/471, 60%), heartburn (63/471, 13%) and chest pain (29/471, 6%) were the most common presenting symptoms. The majority of the patients underwent transhiatal esophagectomy (n = 279, 59.1%) and en-bloc esophagectomy (n = 85, 18.0%). Staging with PET/CT was utilized in 316/471 patients (67%) with 6% of Group 1, 76% of Group 2 and 100% of Group 3, P Conclusion This analysis reveals an improvement in 5-year survival after esophagectomy from 30% to 47% over the past two decades. Similarly, 10-year survival has improved from 23% to 37%. The evolution of better clinical staging and advancements in neoadjuvant therapy likely played a vital role in these trends. In contrast to the earliest cohort, PET/CT is now routinely utilized in the staging of esophageal cancer. Further, other than those with early stage disease, all patients are currently evaluated for neoadjuvant chemo-radiation. Notably, the 5-year survival rate for the most recent cohort (2012–2017) approaches 50% and would likely be higher if patients with esophageal adenocarcinoma treated endoscopically were included. Improvements in staging and treatment paradigms for esophageal adenocarcinoma have resulted in significant progress towards cure. Disclosure All authors have declared no conflicts of interest.

Because of the significant morbidity and lethality caused by alcoholic liver disease (ALD), there remains a need to elucidate the regulatory mechanisms that can be targeted to prevent and treat ALD. Towards this goal, minimally invasive biomarker discovery represents an outstanding approach for these purposes. The mechanisms underlying ALD include hepatic lipid accumulation. As the peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) has been shown to inhibit steatosis, the present study examined the role of PPARβ/δ in ALD coupling metabolomic, biochemical and molecular biological analyses. Wild-type and Pparβ/δ-null mice were fed either a control or 4% ethanol diet and examined after 4–7 months of treatment. Ethanol fed Pparβ/δ-null mice exhibited steatosis after short-term treatment compared to controls, the latter effect appeared to be due to increased activity of sterol regulatory element binding protein 1c (SREBP1c). The wild-type and Pparβ/δ-null mice fed the control diet showed clear differences in their urinary metabolomic profiles. In particular, metabolites associated with arginine and proline metabolism, and glycerolipid metabolism, were markedly different between genotypes suggesting a constitutive role for PPARβ/δ in the metabolism of these amino acids. Interestingly, urinary excretion of taurine was present in ethanol-fed wild-type mice but markedly lower in similarly treated Pparβ/δ-null mice. Evidence suggests that PPARβ/δ modulates pyridoxal kinase activity by altering Km, consistent with the observed decreased in urinary taurine excretion. These data collectively suggest that PPARβ/δ prevents ethanol-induced hepatic effects by inhibiting hepatic lipogenesis, modulation of amino acid metabolism, and altering pyridoxal kinase activity.

The mechanisms underlying perfluorooctane sulfonate (PFOS)-induced steatosis remain unclear. The hypothesis that PFOS causes steatosis and other hepatic effects by forming an ion pair with choline was examined. C57BL/6 mice were fed either a control diet or a marginal methionine/choline-deficient (mMCD) diet, with and without 0.003, 0.006, or 0.012% potassium PFOS. Dietary PFOS caused a dose-dependent decrease in body weight, and increases in the relative liver weight, hepatic triglyceride concentration and serum markers of liver toxicity and oxidative stress. Some of these effects were exacerbated in mice fed the mMCD diet supplemented with 0.012% PFOS compared with those fed the control diet supplemented with 0.012% PFOS. Surprisingly, serum PFOS concentrations were higher while liver PFOS concentrations were lower in mMCD-fed mice compared with corresponding control-fed mice. To determine if supplemental dietary choline could prevent PFOS-induced hepatic effects, C57BL/6 mice were fed a control diet, or a choline supplemental diet (1.2%) with or without 0.003% PFOS. Lipidomic analysis demonstrated that PFOS caused alterations in hepatic lipid metabolism in the PFOS-fed mice compared with controls, and supplemental dietary choline prevented these PFOS-induced changes. Interestingly, dietary choline supplementation also prevented PFOS-induced oxidative damage. These studies are the first to suggest that PFOS may cause hepatic steatosis and oxidative stress by effectively reducing the choline required for hepatic VLDL production and export by forming an ion pair with choline, and suggest that choline supplementation may prevent and/or treat PFOS-induced hepatic steatosis and oxidative stress.