An endogenous molecular clockwork drives various cellular pathways including metabolism and the cell cycle. Its dysregulation is able to prompt pathological phenotypes including cancer. Besides dramatic metabolic alterations, cancer cells display severe changes in the clock phenotype with likely consequences in tumor progression and treatment response. In this study, we use a comprehensive systems-driven approach to investigate the effect of clock disruption on metabolic pathways and its impact on drug response in a cellular model of colon cancer progression. We identified distinctive time-related transcriptomic and metabolic features of a primary tumor and its metastatic counterpart. A mapping of the expression data to a comprehensive genome-scale reconstruction of human metabolism allowed for the in-depth functional characterization of 24 h-oscillating transcripts and pointed to a clock-driven metabolic reprogramming in tumorigenesis. In particular, we identified a set of five clock–regulated glycolysis genes, ALDH3A2, ALDOC, HKDC1, PCK2, and PDHB with differential temporal expression patterns. These findings were validated in organoids and in primary fibroblasts isolated from normal colon and colon adenocarcinoma from the same patient. We further identified a reciprocal connection of HKDC1 to the clock in the primary tumor, which is lost in the metastatic cells. Interestingly, a disruption of the core-clock gene BMAL1 impacts on HKDC1 and leads to a time-dependent rewiring of metabolism, namely an increase in glycolytic activity, as well as changes in treatment response. This work provides novel evidence regarding the complex interplay between the circadian clock and metabolic alterations in carcinogenesis and identifies new connections between both systems with pivotal roles in cancer progression and response to therapy.

Resveratrol has gained tremendous interest owing to multiple reported health-beneficial effects. However, the underlying key mechanism of action of resveratrol remained largely controversial. Here, we demonstrate that under physiologically relevant conditions major biological effects of resveratrol can be attributed to the generation of oxidation products such as reactive oxygen species (ROS). At low hormetic concentrations (< 50 µM), treatment with resveratrol increased cell viability in a set of representative cell models, whereas application of quenchers of ROS completely truncated these beneficial effects. Notably, application of resveratrol led to mild, Nrf2-specific cellular gene expression reprogramming. For example, in 2 primary human epidermal keratinocytes this resulted in a 1.3-fold increase of endogenous metabolites such as glutathione (GSH) and subsequently in a quantitative reduction of the cellular redox environment by 2.61 mV mmol GSH per g protein. After external application of oxidative stress by using 0.8% (v/v) ethanol, endogenous generation of ROS was consequently reduced by 24% in resveratrol pre-treated cells. In contrast to the common perception that resveratrol acts mainly as a chemical antioxidant or as a target protein-specific ligand, we propose that effects from resveratrol treatment are essentially based on oxidative triggering of cells. In relevant physiological microenvironments this effect can lead to hormetic shifting of cellular defence towards a more reductive state to improve resilience to oxidative stress in a manner that can be exactly defined by the redox-environment of the cell.

The mammalian circadian clock and the cell cycle are two major biological oscillators whose coupling influences cell fate decisions. In the present study, we use a model-driven experimental approach to investigate the interplay between clock and cell cycle components and the dysregulatory effects of RAS on this coupled system. In particular, we focus on the Ink4a/Arf locus as one of the bridging clock-cell cycle elements. Upon perturbations by the rat sarcoma viral oncogene (RAS), differential effects on the circadian phenotype were observed in wild-type and Ink4a/Arf knock-out mouse embryonic fibroblasts (MEFs), which could be reproduced by our modelling simulations and correlated with opposing cell cycle fate decisions. Interestingly, the observed changes can be attributed to in silico phase shifts in the expression of core-clock elements. A genome-wide analysis revealed a set of differentially expressed genes that form an intricate network with the circadian system with enriched pathways involved in opposing cell cycle phenotypes. In addition, a machine learning approach complemented by cell cycle analysis classified the observed cell cycle fate decisions as dependent on Ink4a/Arf and the oncogene RAS and highlighted a putative fine-tuning role of Bmal1 as an elicitor of such processes, ultimately resulting in increased cell proliferation in the Ink4a/Arf knock-out scenario. This indicates that the dysregulation of the core-clock might work as an enhancer of RAS-mediated regulation of the cell cycle. Our combined in silico and in vitro approach highlights the important role of the circadian clock as an Ink4a/Arf-dependent modulator of oncogene-induced cell fate decisions, reinforcing its function as a tumour-suppressor and the close interplay between the clock and the cell cycle network.

The circadian clock is a powerful endogenous timing system, which allows organisms to fine-tune their physiology and behaviour to the geophysical time. The interplay of a distinct set of core-clock genes and proteins generates oscillations in expression of output target genes which temporally regulate numerous molecular and cellular processes. The study of the circadian timing at the organismal as well as at the cellular level outlines the field of chronobiology, which has been highly interdisciplinary ever since its origins. The development of high-throughput approaches enables the study of the clock at a systems level. In addition to experimental approaches, computational clock models exist which allow the analysis of rhythmic properties of the clock network. Such mathematical models aid mechanistic understanding and can be used to predict outcomes of distinct perturbations in clock components, thereby generating new hypotheses regarding the putative function of particular clock genes. Perturbations in the circadian timing system are linked to numerous molecular dysfunctions and may result in severe pathologies including cancer. A comprehensive knowledge regarding the mechanistic of the circadian system is crucial to develop new procedures to investigate pathologies associated with a deregulated clock.In this manuscript we review the combination of experimental methodologies, bioinformatics and theoretical models that have been essential to explore this remarkable timing-system. Such an integrative and interdisciplinary approach may provide new strategies with regard to chronotherapeutic treatment and new insights concerning the restoration of the circadian timing in clock-associated diseases.

Amorfrutins are isoprenoid-substituted benzoic acid derivatives, which were found in Amorpha fruticosa L. (bastard indigo) and in Glycyrrhiza foetida Desf. (licorice). Recently, amorfrutins were shown to be selective activators of the nuclear receptor PPARγ. Here, we investigated the effects and PPARγ-based mechanisms of reducing inflammation in colon cells by treatment with amorfrutins. In TNF-α-stimulated colon cells amorfrutin A (1) reduced significantly the expression and secretion of several inflammation mediators, in part due to interaction with PPARγ. These results support the hypothesis that amorfrutins may have the potential to treat inflammation disorders such as chronic inflammatory bowel diseases.

By regulating the timing of cellular processes, the circadian clock provides a way to adapt physiology and behaviour to the geophysical time. In mammals, a light-entrainable master clock located in the suprachiasmatic nucleus (SCN) controls peripheral clocks that are present in virtually every body cell. Defective circadian timing is associated with several pathologies such as cancer and metabolic and sleep disorders. To better understand the circadian regulation of cellular processes, we developed a bioinformatics pipeline encompassing the analysis of high-throughput data sets and the exploitation of published knowledge by text-mining. We identified 118 novel potential clock-regulated genes and integrated them into an existing high-quality circadian network, generating the to-date most comprehensive network of circadian regulated genes (NCRG). To validate particular elements in our network, we assessed publicly available ChIP-seq data for BMAL1, REV-ERBα/β and RORα/γ proteins and found strong evidence for circadian regulation of Elavl1, Nme1, Dhx6, Med1 and Rbbp7 all of which are involved in the regulation of tumourigenesis. Furthermore, we identified Ncl and Ddx6, as targets of RORγ and REV-ERBα, β, respectively. Most interestingly, these genes were also reported to be involved in miRNA regulation; in particular, NCL regulates several miRNAs, all involved in cancer aggressiveness. Thus, NCL represents a novel potential link via which the circadian clock, and specifically RORγ, regulates the expression of miRNAs, with particular consequences in breast cancer progression. Our findings bring us one step forward towards a mechanistic understanding of mammalian circadian regulation, and provide further evidence of the influence of circadian deregulation in cancer.

Accumulating evidence points to a significant role of the circadian clock in the regulation of splicing in various organisms, including mammals. Both dysregulated circadian rhythms and aberrant pre-mRNA splicing are frequently implicated in human disease, in particular in cancer. To investigate the role of the circadian clock in the regulation of splicing in a cancer progression context at the systems-level, we conducted a genome-wide analysis and compared the rhythmic transcriptional profiles of colon carcinoma cell lines SW480 and SW620, derived from primary and metastatic sites of the same patient, respectively. We identified spliceosome components and splicing factors with cell-specific circadian expression patterns including SRSF1, HNRNPLL, ESRP1, and RBM 8A, as well as altered alternative splicing events and circadian alternative splicing patterns of output genes (e.g., VEGFA, NCAM1, FGFR2, CD44) in our cellular model. Our data reveals a remarkable interplay between the circadian clock and pre-mRNA splicing with putative consequences in tumor progression and metastasis.

In the past few years mounting evidences have highlighted the tight correlation between circadian rhythms and metabolism. Although at the organismal level the central timekeeper is constituted by the hypothalamic suprachiasmatic nuclei practically all the peripheral tissues are equipped with autonomous oscillators made up by common molecular clockworks represented by circuits of gene expression that are organized in interconnected positive and negative feed-back loops. In this study we exploited a well-established in vitro synchronization model to investigate specifically the linkage between clock gene expression and the mitochondrial oxidative phosphorylation (OxPhos). Here we show that synchronized cells exhibit an autonomous ultradian mitochondrial respiratory activity which is abrogated by silencing the master clock gene ARNTL/BMAL1. Surprisingly, pharmacological inhibition of the mitochondrial OxPhos system resulted in dramatic deregulation of the rhythmic clock-gene expression and a similar result was attained with mtDNA depleted cells (Rho0). Our findings provide a novel level of complexity in the interlocked feedback loop controlling the interplay between cellular bioenergetics and the molecular clockwork. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.