The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.

Three methylation epigenotypes exist in colorectal cancer, and suitable classification markers have been developed. Intermediate-methylation epigenotype with KRAS-mutation(+) correlated with worse prognosis.

In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.

Small cell lung cancer (SCLC) is a subtype of lung cancer with poor prognosis. Expression array analysis of 23 SCLC cases and 42 normal tissues revealed that EZH2 and other PRC2 members were highly expressed in SCLC. ChIP-seq for H3K27me3 suggested that genes with H3K27me3(+) in SCLC were extended not only to PRC2-target genes in ES cells but also to other target genes such as cellular adhesion-related genes. These H3K27me3(+) genes in SCLC were repressed significantly, and introduction of the most repressed gene JUB into SCLC cell line lead to growth inhibition. Shorter overall survival of clinical SCLC cases correlated to repression of JUB alone, or a set of four genes including H3K27me3(+) genes. Treatment with EZH2 inhibitors, DZNep and GSK126, resulted in growth repression of SCLC cell lines. High PRC2 expression was suggested to contribute to gene repression in SCLC, and may play a role in genesis of SCLC.

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.

Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARID1C. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.

The fibrogenic response in tissue-resident fibroblasts is determined by the balance between activation and repression signals from the tissue microenvironment. While the molecular pathways by which transforming growth factor-1 (TGF-β1) activates pro-fibrogenic mechanisms have been extensively studied and are recognized critical during fibrosis development, the factors regulating TGF-β1 signaling are poorly understood. Here we show that macrophage hypoxia signaling suppresses excessive fibrosis in a heart via oncostatin-m (OSM) secretion. During cardiac remodeling, Ly6C hi monocytes/macrophages accumulate in hypoxic areas through a hypoxia-inducible factor (HIF)-1α dependent manner and suppresses cardiac fibroblast activation. As an underlying molecular mechanism, we identify OSM, part of the interleukin 6 cytokine family, as a HIF-1α target gene, which directly inhibits the TGF-β1 mediated activation of cardiac fibroblasts through extracellular signal-regulated kinase 1/2-dependent phosphorylation of the SMAD linker region. These results demonstrate that macrophage hypoxia signaling regulates fibroblast activation through OSM secretion in vivo.

BACKGROUND: Tumour-initiating cells (TICs) or cancer stem cells can exist as a small population in malignant tissues. The signalling pathways activated in TICs that contribute to tumourigenesis are not fully understood. METHODS: Several breast cancer cell lines were sorted with CD24 and CD44, known markers for enrichment of breast cancer TICs. Tumourigenesis was analysed using sorted cells and total RNA was subjected to gene expression profiling and gene set enrichment analysis (GSEA). RESULTS: We showed that several breast cancer cell lines have a small population of CD24 À/low /CD44 þ cells in which TICs may be enriched, and confirmed the properties of TICs in a xenograft model. GSEA revealed that CD24 À/low /CD44 þ cell populations are enriched for genes involved in transforming growth factor-b, tumour necrosis factor, and interferon response pathways. Moreover, we found the presence of nuclear factor-kB (NF-kB) activity in CD24 À/low /CD44 þ cells, which was previously unrecognised. In addition, NF-kB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumourigenesis of CD24 À/low / CD44 þ cells in vivo. CONCLUSION: Our findings suggest that signalling pathways identified using GSEA help to identify molecular targets and biomarkers for TIC-like cells.