Colon cancer is the third most common cancer and the second leading cause of cancer-related death in the United States, emphasizing the need for the discovery of new cellular targets. Using a metabolomics approach, we report here that epoxygenated fatty acids (EpFA), which are eicosanoid metabolites produced by cytochrome P450 (CYP) monooxygenases, were increased in both the plasma and colon of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon cancer mice. CYP monooxygenases were overexpressed in colon tumor tissues and colon cancer cells. Pharmacologic inhibition or genetic ablation of CYP monooxygenases suppressed AOM/DSS-induced colon tumorigenesis in vivo. In addition, treatment with 12,13-epoxyoctadecenoic acid (EpOME), which is a metabolite of CYP monooxygenase produced from linoleic acid, increased cytokine production and JNK phosphorylation in vitro and exacerbated AOM/DSS-induced colon tumorigenesis in vivo.Together, these results demonstrate that the previously unappreciated CYP monooxygenase pathway is upregulated in colon cancer, contributes to its pathogenesis, and could be therapeutically explored for preventing or treating colon cancer.Significance: This study finds that the previously unappreciated CYP monooxygenase eicosanoid pathway is deregulated in colon cancer and contributes to colon tumorigenesis. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Figure 4. EpOME exaggerates AOM/DSS-induced colon tumorigenesis in vivo. A, Scheme of animal experiment to test the effect of 12,13-EpOME (dose, 2 mg/kg/day; administered via mini-pump) on colon tumorigenesis. B, Quantification of colon tumorigenesis in mice (n ¼ 8-9 per group). C, Expression of proinflammatory and protumorigenic genes in colon (n ¼ 6-8 per group). D, Quantification of CD45 þ and CD45 þ F4/80 þ immune cells in colon (n ¼ 7-8 per group). E, Hematoxylin and eosin (H&E) histology and IHC staining of PCNA and b-catenin in colon (n ¼ 6-7 per group; scale bar, 50 mm). The results are expressed as mean AE SEM. The statistical significance of two groups was determined using Student t test or Wilcoxon-Mann test.
1The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables 2 in providing tight control over many biological paradigms. There are clear roles for 3 branched architectures in regulating proteasome-mediated degradation, however the 4 proteins that selectively recognize and process these atypical chains are unknown. Here, 5 using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, 6 we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, exclusively 7 cleaves K48 branched chains. The activity and selectivity toward branched chains is 8 markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using proteasome 9 complexes reconstituted with either active or inactive UCH37 together with protein 10 substrates modified with branched chains, we find that chain debranching promotes 11 degradation under multi-turnover conditions. These results are further supported by 12 proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity 13 impairs global protein turnover. Our work therefore defines UCH37 as a debranching 14 deubiquitinase important for promoting proteasomal degradation.
Circadian rhythms are essential for controlling the cell cycle, cellular proliferation, and apoptosis, and hence, are tightly linked to cell fate. Disruption of circadian rhythms has been shown to trigger various pathological developments, including cancer. Several recent studies have used a variety of small molecules to affect circadian oscillations, however, their concomitant cellular effects were not assessed. Here, we use five molecules, grouped into direct versus indirect effectors of the circadian clock, to modulate periods in a human osteosarcoma cell line (U2OS), and determined their influences on cellular behaviors, including motility and colony formation. Luciferase reporters, whose expression were driven via Bmal1and Per2-promoters (positive and negative protein components of the core clock), were used to facilitate the visualization and quantitative analysis of circadian oscillations. We show that all molecules significantly increase or decrease the circadian periods of Bmal1 and Per2 in a dosedependent manner, but period length does not correlate with the extent of cell migration or proliferation. We observed that only molecules that affected circadian oscillations to a greater extent showed significant influence on cell functions (e.g. motility and colony formation).Because it is important to consider the likelihood of biological effects resulting from noncircadian targets, we also provide a thorough discussion of potential modes of action. Future studies should employ additional compounds that directly target circadian proteins and/or have different circadian effects, and evaluation in other cancer models to determine whether results obtained here remain consistent. proteins aryl hydrocarbon receptor nuclear translocator-like protein 1 (ARNTL/BMAL1) and circadian locomotor output cycles kaput (CLOCK) heterodimerize in the morning and bind to the E-box promoter, which initiates the expression of Period (Per) and Cryptochrome (Cry). PER and CRY accumulate in the evening and form a heterodimer, which translocates back into the nucleus and inhibits Bmal1 and Clock activity. 18 As a result, the core circadian machinery oscillates with a period of approximately 24 h. In cell culture, intrinsic and self-sustained circadian clocks are persistent even in the absence of external time cues. 19 To synchronize clocks in cultured cells, treatments of high concentration serum 20 or chemical reagents (e.g. dexamethasone 21 or forskolin 22 ) are frequently used. Circadian rhythmicity is tightly associated with post-translational modifications of clock proteins. 23 Phosphorylation of most clock proteins occurs in a rhythmic manner; thus, alteration of clock protein phosphorylation can result in changes to circadian periods. 24 Genetic manipulations of the post-translational regulators of clock proteins have been shown to affect circadian functions and periodicity. 24-25 However, conventional genetic approaches also result in fatality, pleiotropy, and functional redundancy. Alternatively, chemical modulation of these...
Circadian rhythms are essential for controlling the cell cycle, cellular proliferation, and apoptosis, and hence are tightly linked to cell fate. Several recent studies have used small molecules to affect circadian oscillations; however, their concomitant cellular effects were not assessed, and they have not been compared under similar experimental conditions. In this work, we use five molecules, grouped into direct versus indirect effectors of the circadian clock, to modulate periods in a human osteosarcoma cell line (U2OS) and determine their influences on cellular behaviors, including motility and colony formation. Luciferase reporters, whose expression was driven via Bmal1- or Per2-promoters, were used to facilitate the visualization and quantitative analysis of circadian oscillations. We show that all molecules increase or decrease the circadian periods of Bmal1 and Per2 in a dose-dependent manner, but period length does not correlate with the extent of cell migration or proliferation. Nonetheless, molecules that affected circadian oscillations to a greater degree resulted in substantial influence on cellular behaviors (ie, motility and colony formation), which may also be attributable to noncircadian targets. Furthermore, we find that the ability and extent to which the molecules are able to affect oscillations is independent of whether they are direct or indirect modulators. Because of the numerous connections and feedback between the circadian clock and other pathways, it is important to consider the effects of both in assessing these and other compounds.
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