Detection of microbial components such as lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) on macrophages induces a robust pro-inflammatory response that is dependent on metabolic reprogramming. These innate metabolic changes have been compared to aerobic glycolysis in tumour cells. However, the mechanisms by which TLR4 activation leads to mitochondrial and glycolytic reprogramming are unknown. Here we show that TLR4 activation induces a signalling cascade recruiting TRAF6 and TBK-1, while TBK-1 phosphorylates STAT3 on S727. Using a genetically engineered mouse model incapable of undergoing STAT3 Ser727 phosphorylation, we show ex vivo and in vivo that STAT3 Ser727 phosphorylation is critical for LPS-induced glycolytic reprogramming, production of the central immune response metabolite succinate and inflammatory cytokine production in a model of LPS-induced inflammation. Our study identifies non-canonical STAT3 activation as the crucial signalling intermediary for TLR4-induced glycolysis, macrophage metabolic reprogramming and inflammation.

Hedgehog (Hh) signaling regulates cell fate and self-renewal in development and cancer. Canonical Hh signaling is mediated by Hh ligand binding to the receptor Patched (Ptch), which in turn activates Gli-mediated transcription through Smoothened (Smo), the molecular target of the Hh pathway inhibitors used as cancer therapeutics. Small cell lung cancer (SCLC) is a common, aggressive malignancy with universally poor prognosis. Although preclinical studies have shown that Hh inhibitors block the self-renewal capacity of SCLC cells, the lack of activating pathway mutations have cast doubt over the significance of these observations. In particular, the existence of autocrine, ligand-dependent Hh signaling in SCLC has been disputed. In a conditional Tp53;Rb1 mutant mouse model of SCLC, we now demonstrate a requirement for the Hh ligand Sonic Hedgehog (Shh) for the progression of SCLC. Conversely, we show that conditional Shh overexpression activates canonical Hh signaling in SCLC cells, and markedly accelerates tumor progression. When compared to mouse SCLC tumors expressing an activating, ligand-independent Smo mutant, tumors overexpressing Shh exhibited marked chromosomal instability and Smoothened-independent upregulation of Cyclin B1, a putative non-canonical arm of the Hh pathway. In turn, we show that overexpression of Cyclin B1 induces chromosomal instability in mouse embryonic fibroblasts lacking both Tp53 and Rb1. These results provide strong support for an autocrine, ligand-dependent model of Hh signaling in SCLC pathogenesis, and reveal a novel role for non-canonical Hh signaling through the induction of chromosomal instability.

BackgroundDifferent cell types possess different copies of mtDNA to support their specific requirements for cellular metabolism. Cell-specific mtDNA copy numbers are established through cell-specific mtDNA replication during cell differentiation. However, cancer cells are trapped in a “pseudo-differentiated” state as they fail to expand mtDNA copy number. Global DNA methylation can regulate this process, as induced DNA demethylation promotes differentiation of cancer cells and expansion of mtDNA copy number.ResultsTo determine the role that mtDNA methylation plays in regulating mtDNA replication during tumorigenesis, we have characterized the patterns of mtDNA methylation using glioblastoma and osteosarcoma tumor models that have different combinations of mtDNA genotypes and copy number against common nuclear genome backgrounds at different stages of tumor progression. To ensure the reliability of the findings, we have applied a robust experimental pipeline including three approaches, namely whole-mtDNA bisulfite-sequencing with mtDNA-genotype-specific analysis, pyrosequencing, and methylated immunoprecipitation against 5mC and 5hmC. We have determined genotype-specific methylation profiles, which were modulated through tumor progression. Moreover, a strong influence from the nuclear genome was also observed on mtDNA methylation patterns using the same mtDNA genotype under different nuclear genomes. Furthermore, the numbers of mtDNA copy in tumor-initiating cells affected mtDNA methylation levels in late-stage tumors.ConclusionsOur findings highlight the influences that the nuclear and mitochondrial genomes have in setting mtDNA methylation patterns to regulate mtDNA copy number in tumorigenesis. They have important implications for assessing global DNA methylation patterns in tumorigenesis and the availability of mtDNA template for mtDNA replication.Electronic supplementary materialThe online version of this article (10.1186/s13148-018-0590-0) contains supplementary material, which is available to authorized users.