Molecular characterization of the circadian clock in patients with Parkinson's disease-CLOCK4PD Study protocol.

INTRODUCTION: Circadian rhythms (CRs) orchestrate intrinsic 24-hour oscillations which synchronize an organism's physiology and behaviour with respect to daily cycles. CR disruptions have been linked to Parkinson's Disease (PD), the second most prevalent neurodegenerative disorder globally, and are associated to several PD-symptoms such as sleep disturbances. Studying molecular changes of CR offers a potential avenue for unravelling novel insights into the PD progression, symptoms, and can be further used for optimization of treatment strategies. Yet, a comprehensive characterization of the alterations at the molecular expression level for core-clock and clock-controlled genes in PD is still missing. METHODS AND ANALYSIS: The proposed study protocol will be used to characterize expression profiles of circadian genes obtained from saliva samples in PD patients and controls. For this purpose, 20 healthy controls and 70 PD patients will be recruited. Data from clinical assessment, questionnaires, actigraphy tracking and polysomnography will be collected and clinical evaluations will be repeated as a follow-up in one-year time. We plan to carry out sub-group analyses considering several clinical factors (e.g., biological sex, treatment dosages, or fluctuation of symptoms), and to correlate reflected changes in CR of measured genes with distinct PD phenotypes (diffuse malignant and mild/motor-predominant). Additionally, using NanoStringⓇ multiplex technology on a subset of samples, we aim to further explore potential CR alterations in hundreds of genes involved in neuropathology pathways. DISCUSSION: CLOCK4PD is a mono-centric, non-interventional observational study aiming at the molecular characterization of CR alterations in PD. We further plan to determine physiological modifications in sleep and activity patterns, and clinical factors correlating with the observed CR changes. Our study may provide valuable insights into the intricate interplay between CR and PD with a potential to be used as a predictor of circadian alterations reflecting distinct disease phenotypes, symptoms, and progression outcomes.

Molecular mechanisms of tumour development in glioblastoma: an emerging role for the circadian clock.

Glioblastoma is one of the most lethal cancers with current therapeutic options lacking major successes. This underlines the necessity to understand glioblastoma biology on other levels and use these learnings for the development of new therapeutic concepts. Mounting evidence in the field of circadian medicine points to a tight interplay between disturbances of the circadian system and glioblastoma progression. The circadian clock, an internal biological mechanism governing numerous physiological processes across a 24-h cycle, also plays a pivotal role in regulationg key cellular functions, including DNA repair, cell cycle progression, and apoptosis. These processes are integral to tumour development and response to therapy. Disruptions in circadian rhythms can influence tumour growth, invasion, and response to treatment in glioblastoma patients. In this review, we explore the robust association between the circadian clock, and cancer hallmarks within the context of glioblastoma. We further discuss the impact of the circadian clock on eight cancer hallmarks shown previously to link the molecular clock to different cancers, and summarize the putative role of clock proteins in circadian rhythm disturbances and chronotherapy in glioblastoma. By unravelling the molecular mechanisms behind the intricate connections between the circadian clock and glioblastoma progression, researchers can pave the way for the identification of potential therapeutic targets, the development of innovative treatment strategies and personalized medicine approaches. In conclusion, this review underscores the significant influence of the circadian clock on the advancement and understanding of future therapies in glioblastoma, ultimately leading to enhanced outcomes for glioblastoma patients.

The circadian clock circuitry modulates leukemia initiating cell activity in T-cell acute lymphoblastic leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy, characterized by restricted cellular subsets with asymmetrically enriched leukemia initiating cell (LIC) activity. Nonetheless, it is still unclear which signaling programs promote LIC maintenance and progression. METHODS: Here, we evaluated the role of the biological clock in the regulation of the molecular mechanisms and signaling pathways impacting the cellular dynamics in T-ALL through an integrated experimental approach including gene expression profiling of shRNA-modified T-ALL cell lines and Chromatin Immunoprecipitation Sequencing (ChIP-Seq) of leukemic cells. Patient-derived xenograft (PDXs) cell subsets were also genetically manipulated in order to assess the LIC activity modulated by the loss of biological clock in human T-ALL. RESULTS: We report that the disruption of the circadian clock circuitry obtained through shRNA-mediated knockdown of CLOCK and BMAL1 genes negatively impacted the growth in vitro as well as the activity in vivo of LIC derived from PDXs after transplantation into immunodeficient recipient mice. Additionally, gene expression data integrated with ChIP-Seq profiles of leukemic cells revealed that the circadian clock directly promotes the expression of genes, such as IL20RB, crucially involved in JAK/STAT signaling, making the T-ALL cells more responsive to Interleukin 20 (IL20). CONCLUSION: Taken together, our data support the concept that the biological clock drives the expression of IL20R prompting JAK/STAT signaling and promoting LIC activity in T-ALL and suggest that the selective targeting of circadian components could be therapeutically relevant for the treatment of T-ALL patients.

Differential expression of the circadian clock network correlates with tumour progression in gliomas.

BACKGROUND: Gliomas are tumours arising mostly from astrocytic or oligodendrocytic precursor cells. These tumours are classified according to the updated WHO classification from 2021 in 4 grades depending on molecular and histopathological criteria. Despite novel multimodal therapeutic approaches, the vast majority of gliomas (WHO grade III and IV) are not curable. The circadian clock is an important regulator of numerous cellular processes and its dysregulation had been found during the progression of many cancers, including gliomas. RESULTS: In this study, we explore expression patterns of clock-controlled genes in low-grade glioma (LGG) and glioblastoma multiforme (GBM) and show that a set of 45 clock-controlled genes can be used to distinguish GBM from normal tissue. Subsequent analysis identified 17 clock-controlled genes with a significant association with survival. The results point to a loss of correlation strength within elements of the circadian clock network in GBM compared to LGG. We further explored the progression patterns of mutations in LGG and GBM, and showed that tumour suppressor APC is lost late both in LGG and GBM. Moreover, HIF1A, involved in cellular response to hypoxia, exhibits subclonal losses in LGG, and TERT, involved in the formation of telomerase, is lost late in the GBM progression. By examining multi-sample LGG data, we find that the clock-controlled driver genes APC, HIF1A, TERT and TP53 experience frequent subclonal gains and losses. CONCLUSIONS: Our results show a higher level of disrgulation at the gene expression level in GBM compared to LGG, and indicate an association between the differentially expressed clock-regulated genes and patient survival in both LGG and GBM. By reconstructing the patterns of progression in LGG and GBM, our data reveals the relatively late gains and losses of clock-regulated glioma drivers. Our analysis emphasizes the role of clock-regulated genes in glioma development and progression. Yet, further research is needed to asses their value in the development of new treatments.

An integrative mathematical model for timing treatment toxicity and Zeitgeber impact in colorectal cancer cells.

Increasing evidence points to a role of the circadian clock in the regulation of cancer hallmarks with a strong impact on the understanding and treatment of this disease. Anti-cancer treatment can be personalized considering treatment timing. Here we present a new mathematical model based on data from three colorectal cancer cell lines and core-clock knock-outs, which couples the circadian and drug metabolism network, and that allows to determine toxicity profiles for a given drug and cell type. Moreover, this model integrates external Zeitgebers and thus may be used to fine-tune toxicity by using external factors, such as light, and therefore, to a certain extent, help fitting the endogenous rhythms of the patients to a defined clinic routine facilitating the implementation of time-dependent treatment in clinical practice.

TimeTeller for timing health: The potential of circadian medicine to improve performance, prevent disease and optimize treatment.

Circadian medicine, the study of the effects of time on health and disease has seen an uprising in recent years as a means to enhance health and performance, and optimize treatment timing. Our endogenous time generating system -the circadian clock- regulates behavioural, physiological and cellular processes. Disruptions of the clock, via external factors like shift work or jet lag, or internal perturbations such as genetic alterations, are linked to an increased risk of various diseases like obesity, diabetes, cardiovascular diseases and cancer. By aligning an individual's circadian clock with optimal times for performing daily routines, physical and mental performance, and also the effectiveness of certain therapies can be improved. Despite the benefits of circadian medicine, the lack of non-invasive tools for characterizing the clock limits the potential of the field. TimeTeller is a non-invasive molecular/digital tool for the characterization of circadian rhythms and prediction of daily routines, including treatment timing, to unlock the potential of circadian medicine and implementing it in various settings. Given the multiple known and potentially yet unknown dependent health factors of individual circadian rhythms, the utility of this emerging biomarker is best exploited in data driven, personalized medicine use cases, using health information across lifestyle, care, and research settings.

Molecular characterization of the circadian clock in paediatric leukaemia patients: a prospective study protocol.

BACKGROUND: In many organisms, including humans, the timing of cellular processes is regulated by the circadian clock. At the molecular level the core-clock consists of transcriptional-translational-feedback loops including several genes such as BMAL1, CLOCK, PERs and CRYs generating circa 24-h rhythms in the expression of about 40% of our genes across all tissues. Previously these core-clock genes have been shown to be differentially expressed in various cancers. Albeit a significant effect in treatment optimization of chemotherapy timing in paediatric acute lymphoblastic leukaemia has previously been reported, the mechanistic role played by the molecular circadian clock in acute paediatric leukaemia remains elusive. METHODS: To characterize the circadian clock, we will recruit patients with newly diagnosed leukaemia and collect time course saliva and blood samples, as well as a single bone marrow sample. From the blood and bone marrow samples nucleated cells will be isolated and further undergo separation into CD19+ and CD19- cells. qPCR is performed on all samples targeting the core-clock genes including BMAL1, CLOCK, PER2 and CRY1. Resulting data will be analysed for circadian rhythmicity using the RAIN algorithm and harmonic regression. DISCUSSION: To the best of our knowledge this is the first study aiming to characterize the circadian clock in a cohort of paediatric patients with acute leukaemia. In the future we hope to contribute to uncovering further vulnerabilities of cancers associated with the molecular circadian clock and in particular adjust chemotherapy accordingly, leading to more targeted toxicity, and hence decreased systemic toxicities.

Chronotherapy in Glioblastoma: state of the art and future perspectives.

Circadian rhythms regulate various processes in the human body, including drug metabolism. Chronotherapy optimizes treatment timing based on the circadian rhythm of the individual patient, such that the treatment efficacy is maximized, and adverse effects are minimized. It has been explored in different cancers with varying conclusions. Glioblastoma multiforme (GBM) is the most aggressive type of brain tumour with a very dismal prognosis. In recent years, there has been very little success in designing successful therapies to fight this disease. Chronotherapy offers the opportunity to leverage existing treatments to extend patient survival and to increase their quality of life. Here, we discuss recent advances in using chronotherapy regimens in the treatment of GMB, such as radiotherapy, temozolomide (TMZ) and bortezomib, as well as discuss novel treatments with drugs of short half-life or circadian phase specific activity, and examine the therapeutic potential of new approaches that target elements of the core circadian clock.

Antiproliferative Effects of Cynara Cardunculus in Colorectal Cancer Cells Are Modulated by the Circadian Clock.

The circadian clock generates 24 h rhythms in behavioural, cellular and molecular processes. Malfunctions of the clock are associated with enhanced susceptibility to cancer, worse treatment response and poor prognosis. Clock-controlled genes are involved in cellular processes associated with tumour development and progression including metabolism of drugs and the cell cycle. Cynara cardunculus, a plant of the Asteraceae family, has been reported to have antiproliferative effects on breast cancer cells. Here, we used the human colorectal cancer (CRC) cell line HCT116 and its knockout variants for different core-clock genes (BMAL1, PER2, NR1D1), to investigate the treatment effect of C. cardunculus lipophilic leaf extract under different clock scenarios. Our results show a direct effect of C. cardunculus on the circadian phenotype of the cells, as indicated by alterations in the phase, amplitude, and period length of core-clock gene oscillations. Furthermore, our data indicate a role for the circadian clock in sensitivity to C. cardunculus treatment. In particular, the treatment inhibited proliferation and induced cytotoxicity and apoptosis in a clock knockout-specific manner, in CRC cells. These results point to a potential effect of C. cardunculus lipophilic leaf extracts as a modulator of the circadian clock, in addition to its anti-proliferative properties.

Core-Clock Genes Regulate Proliferation and Invasion via a Reciprocal Interplay with MACC1 in Colorectal Cancer Cells.

The circadian clock coordinates the timing of several cellular processes including transcription, the cell cycle, and metabolism. Disruptions in the clock machinery trigger the abnormal regulation of cancer hallmarks, impair cellular homeostasis, and stimulate tumourigenesis. Here we investigated the role of a disrupted clock by knocking out or knocking down the core-clock (CC) genes ARNTL, PER2 or NR1D1 in cancer progression (e.g., cell proliferation and invasion) using colorectal cancer (CRC) cell lines HCT116, SW480 and SW620, from different progression stages with distinct clock phenotypes, and identified mechanistic links from the clock to altered cancer-promoting cellular properties. We identified MACC1 (metastasis-associated in colon cancer 1), a known driver for metastasis and an EMT (epithelial-to-mesenchymal transition)-related gene, to be significantly differentially expressed in CC manipulated cells and analysed the effect of MACC1 manipulation (knockout or overexpression) in terms of circadian clock phenotype as well as cancer progression. Our data points to a bi-directional MACC1-circadian clock interplay in CRC, via CC genes. In particular, knocking out MACC1 reduced the period of oscillations, while its overexpression increased it. Interestingly, we found the MACC1 protein to be circadian expressed in HCT116 WT cells, which was disrupted after the knockout of CC genes, and identified a MACC1-NR1D1 protein-protein interaction. In addition, MACC1 manipulation and CC knockout altered cell invasion properties of HCT116 cells, pointing to a regulation of clock and cancer progression in CRC, possibly via the interaction of MACC1 with core-clock genes.

It's About Time: The Circadian Network as Time-Keeper for Cognitive Functioning, Locomotor Activity and Mental Health.

A variety of organisms including mammals have evolved a 24h, self-sustained timekeeping machinery known as the circadian clock (biological clock), which enables to anticipate, respond, and adapt to environmental influences such as the daily light and dark cycles. Proper functioning of the clock plays a pivotal role in the temporal regulation of a wide range of cellular, physiological, and behavioural processes. The disruption of circadian rhythms was found to be associated with the onset and progression of several pathologies including sleep and mental disorders, cancer, and neurodegeneration. Thus, the role of the circadian clock in health and disease, and its clinical applications, have gained increasing attention, but the exact mechanisms underlying temporal regulation require further work and the integration of evidence from different research fields. In this review, we address the current knowledge regarding the functioning of molecular circuits as generators of circadian rhythms and the essential role of circadian synchrony in a healthy organism. In particular, we discuss the role of circadian regulation in the context of behaviour and cognitive functioning, delineating how the loss of this tight interplay is linked to pathological development with a focus on mental disorders and neurodegeneration. We further describe emerging new aspects on the link between the circadian clock and physical exercise-induced cognitive functioning, and its current usage as circadian activator with a positive impact in delaying the progression of certain pathologies including neurodegeneration and brain-related disorders. Finally, we discuss recent epidemiological evidence pointing to an important role of the circadian clock in mental health.

Transcriptome analysis of clock disrupted cancer cells reveals differential alternative splicing of cancer hallmarks genes.

Emerging evidence points towards a regulatory role of the circadian clock in alternative splicing (AS). Whether alterations in core-clock components may contribute to differential AS events is largely unknown. To address this, we carried out a computational analysis on recently generated time-series RNA-seq datasets from three core-clock knockout (KO) genes (ARNTL, NR1D1, PER2) and WT of a colorectal cancer (CRC) cell line, and time-series RNA-seq datasets for additional CRC and Hodgkin's lymphoma (HL) cells, murine WT, Arntl KO, and Nr1d1/2 KO, and murine SCN WT tissue. The deletion of individual core-clock genes resulted in the loss of circadian expression in crucial spliceosome components such as SF3A1 (in ARNTLKO), SNW1 (in NR1D1KO), and HNRNPC (in PER2KO), which led to a differential pattern of KO-specific AS events. All HCT116KO cells showed a rhythmicity loss of a crucial spliceosome gene U2AF1, which was also not rhythmic in higher progression stage CRC and HL cancer cells. AS analysis revealed an increase in alternative first exon events specific to PER2 and NR1D1 KO in HCT116 cells, and a KO-specific change in expression and rhythmicity pattern of AS transcripts related to cancer hallmarks genes including FGFR2 in HCT116_ARNTLKO, CD44 in HCT116_NR1D1KO, and MET in HCT116_PER2KO. KO-specific changes in rhythmic properties of known spliced variants of these genes (e.g. FGFR2 IIIb/FGFR2 IIIc) correlated with epithelial-mesenchymal-transition signalling. Altogether, our bioinformatic analysis highlights a role for the circadian clock in the regulation of AS, and reveals a potential impact of clock disruption in aberrant splicing in cancer hallmark genes.

A Computational Analysis in a Cohort of Parkinson's Disease Patients and Clock-Modified Colorectal Cancer Cells Reveals Common Expression Alterations in Clock-Regulated Genes.

Increasing evidence suggests a role for circadian dysregulation in prompting disease-related phenotypes in mammals. Cancer and neurodegenerative disorders are two aging related diseases reported to be associated with circadian disruption. In this study, we investigated a possible effect of circadian disruption in Parkinson's disease (PD) and colorectal cancer (CRC). We used high-throughput data sets retrieved from whole blood of idiopathic PD (IPD) patients and time course data sets derived from an in vitro model of CRC including the wildtype and three core-clock knockout (KO) cell lines. Several gene expression alterations in IPD patients resembled the expression profiles in the core-clock KO cells. These include expression changes in DBP, GBA, TEF, SNCA, SERPINA1 and TGFB1. Notably, our results pointed to alterations in the core-clock network in IPD patients when compared to healthy controls and revealed variations in the expression profile of PD-associated genes (e.g., HRAS and GBA) upon disruption of the core-clock genes. Our study characterizes changes at the transcriptomic level following circadian clock disruption on common cellular pathways associated with cancer and neurodegeneration (e.g., immune system, energy metabolism and RNA processing), and it points to a significant influence on the overall survival of colon cancer patients for several genes resulting from our analysis (e.g., TUBB6, PAK6, SLC11A1).

A mathematical model of the circadian clock and drug pharmacology to optimize irinotecan administration timing in colorectal cancer.

Scheduling anticancer drug administration over 24 h may critically impact treatment success in a patient-specific manner. Here, we address personalization of treatment timing using a novel mathematical model of irinotecan cellular pharmacokinetics and -dynamics linked to a representation of the core clock and predict treatment toxicity in a colorectal cancer (CRC) cellular model. The mathematical model is fitted to three different scenarios: mouse liver, where the drug metabolism mainly occurs, and two human colorectal cancer cell lines representing an in vitro experimental system for human colorectal cancer progression. Our model successfully recapitulates quantitative circadian datasets of mRNA and protein expression together with timing-dependent irinotecan cytotoxicity data. The model also discriminates time-dependent toxicity between the different cells, suggesting that treatment can be optimized according to their cellular clock. Our results show that the time-dependent degradation of the protein mediating irinotecan activation, as well as an oscillation in the death rate may play an important role in the circadian variations of drug toxicity. In the future, this model can be used to support personalized treatment scheduling by predicting optimal drug timing based on the patient's gene expression profile.

Long-term continuous positive airway pressure treatment ameliorates biological clock disruptions in obstructive sleep apnea.

BACKGROUND: Obstructive Sleep Apnea (OSA) is a highly prevalent and underdiagnosed sleep disorder. Recent studies suggest that OSA might disrupt the biological clock, potentially causing or worsening OSA-associated comorbidities. However, the effect of OSA treatment on clock disruption is not fully understood. METHODS: The impact of OSA and short- (four months) and long-term (two years) OSA treatment, with Continuous Positive Airway Pressure (CPAP), on the biological clock was investigated at four time points within 24 h, in OSA patients relative to controls subjects (no OSA) of the same sex and age group, in a case-control study. Plasma melatonin and cortisol, body temperature and the expression levels and rhythmicity of eleven clock genes in peripheral blood mononuclear cells (PBMCs) were assessed. Additional computational tools were used for a detailed data analysis. FINDINGS: OSA impacts on clock outputs and on the expression of several clock genes in PBMCs. Neither short- nor long-term treatment fully reverted OSA-induced alterations in the expression of clock genes. However, long-term treatment was able to re-establish levels of plasma melatonin and cortisol and body temperature. Machine learning methods could discriminate controls from untreated OSA patients. Following long-term treatment, the distinction between controls and patients disappeared, suggesting a closer similarity of the phenotypes. INTERPRETATION: OSA alters biological clock-related characteristics that differentially respond to short- and long-term CPAP treatment. Long-term CPAP was more efficient in counteracting OSA impact on the clock, but the obtained results suggest that it is not fully effective. A better understanding of the impact of OSA and OSA treatment on the clock may open new avenues to OSA diagnosis, monitoring and treatment.

An Optimal Time for Treatment-Predicting Circadian Time by Machine Learning and Mathematical Modelling.

Tailoring medical interventions to a particular patient and pathology has been termed personalized medicine. The outcome of cancer treatments is improved when the intervention is timed in accordance with the patient's internal time. Yet, one challenge of personalized medicine is how to consider the biological time of the patient. Prerequisite for this so-called chronotherapy is an accurate characterization of the internal circadian time of the patient. As an alternative to time-consuming measurements in a sleep-laboratory, recent studies in chronobiology predict circadian time by applying machine learning approaches and mathematical modelling to easier accessible observables such as gene expression. Embedding these results into the mathematical dynamics between clock and cancer in mammals, we review the precision of predictions and the potential usage with respect to cancer treatment and discuss whether the patient's internal time and circadian observables, may provide an additional indication for individualized treatment timing. Besides the health improvement, timing treatment may imply financial advantages, by ameliorating side effects of treatments, thus reducing costs. Summarizing the advances of recent years, this review brings together the current clinical standard for measuring biological time, the general assessment of circadian rhythmicity, the usage of rhythmic variables to predict biological time and models of circadian rhythmicity.

Circadian Dysregulation of the TGFß/SMAD4 Pathway Modulates Metastatic Properties and Cell Fate Decisions in Pancreatic Cancer Cells.

Impairment of circadian rhythms impacts carcinogenesis. SMAD4, a clock-controlled gene and central component of the TGFß canonical pathway, is frequently mutated in pancreatic ductal adenocarcinoma (PDA), leading to decreased survival. Here, we used an in vitro PDA model of SMAD4-positive and SMAD4-negative cells to investigate the interplay between circadian rhythms, the TGFß canonical signaling pathway, and its impact on tumor malignancy. Our data show that TGFß1, SMAD3, SMAD4, and SMAD7 oscillate in a circadian fashion in SMAD4-positive PDA cells, whereas altering the clock impairs the mRNA dynamics of these genes. Furthermore, the expression of the clock genes DEC1, DEC2, and CRY1 varied depending on SMAD4 status. TGFß pathway activation resulted in an altered clock, cell-cycle arrest, accelerated apoptosis rate, enhanced invasiveness, and chemosensitivity. Our data suggest that the impact of TGFß on the clock is SMAD4-dependent, and S MAD3, SMAD4, DEC1, and CRY1 involved in this cross-talk affect PDA patient survival.

Analysis of the Circadian Regulation of Cancer Hallmarks by a Cross-Platform Study of Colorectal Cancer Time-Series Data Reveals an Association with Genes Involved in Huntington's Disease.

Accumulating evidence points to a link between circadian clock dysfunction and the molecular events that drive tumorigenesis. Here, we investigated the connection between the circadian clock and the hallmarks of cancer in an in vitro model of colorectal cancer (CRC). We used a cross-platform data normalization method to concatenate and compare available microarray and RNA-sequencing time series data of CRC cell lines derived from the same patient at different disease stages. Our data analysis suggests differential regulation of molecular pathways between the CRC cells and identifies several of the circadian and likely clock-controlled genes (CCGs) as cancer hallmarks and circadian drug targets. Notably, we found links of the CCGs to Huntington's disease (HD) in the metastasis-derived cells. We then investigated the impact of perturbations of our candidate genes in a cohort of 439 patients with colon adenocarcinoma retrieved from the Cancer Genome Atlas (TCGA). The analysis revealed a correlation of the differential expression levels of the candidate genes with the survival of patients. Thus, our study provides a bioinformatics workflow that allows for a comprehensive analysis of circadian properties at different stages of colorectal cancer, and identifies a new association between cancer and HD.

Effect of naive and cancer-educated fibroblasts on colon cancer cell circadian growth rhythm.

Opportunistic modification of the tumour microenvironment by cancer cells enhances tumour expansion and consequently eliminates tumour suppressor components. We studied the effect of fibroblasts on the circadian rhythm of growth and protein expression in colon cancer HCT116 cells and found diminished oscillation in the proliferation of HCT116 cells co-cultured with naive fibroblasts, compared with those co-cultured with tumour-associated fibroblasts (TAFs) or those cultured alone, suggesting that TAFs may have lost or gained factors that regulate circadian phenotypes. Based on the fibroblast paracrine factor analysis, we tested IL6, which diminished HCT116 cell growth oscillation, inhibited early phase cell proliferation, increased early phase expression of the differentiation markers CEA and CDX2, and decreased early phase ERK5 phosphorylation. In conclusion, our data demonstrate how the cancer education of naive fibroblasts influences the circadian parameters of neighbouring cancer cells and highlights a putative role for IL6 as a novel candidate for preoperative treatments.

The Core-Clock Gene NR1D1 Impacts Cell Motility In Vitro and Invasiveness in A Zebrafish Xenograft Colon Cancer Model.

Malfunctions of circadian clock trigger abnormal cellular processes and influence tumorigenesis. Using an in vitro and in vivo xenograft model, we show that circadian clock disruption via the downregulation of the core-clock genes BMAL1, PER2, and NR1D1 impacts the circadian phenotype of MYC, WEE1, and TP53, and affects proliferation, apoptosis, and cell migration. In particular, both our in vitro and in vivo results suggest an impairment of cell motility and a reduction in micrometastasis formation upon knockdown of NR1D1, accompanied by altered expression levels of SNAI1 and CD44. Interestingly we show that differential proliferation and reduced tumour growth in vivo may be due to the additional influence of the host-clock and/or to the 3D tumour architecture. Our results raise new questions concerning host-tumour interaction and show that core-clock genes are involved in key cancer properties, including the regulation of cell migration and invasion by NR1D1 in zebrafish xenografts.