BackgroundInterferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. MethodsIn this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. ResultsNovel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. ConclusionsOur results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.
Epithelial to mesenchymal transition (EMT) is a dynamic process that drives cancer cell plasticity and is thought to play a major role in metastasis. Here we show, using MDA-MB-231 cells as a model, that the plasticity of at least some metastatic breast cancer cells is dependent on the transcriptional co-regulator CBFβ. We demonstrate that CBFβ is essential to maintain the mesenchymal phenotype of triple-negative breast cancer cells and that CBFβ-depleted cells undergo a mesenchymal to epithelial transition (MET) and re-organise into acini-like structures, reminiscent of those formed by epithelial breast cells. We subsequently show, using an inducible CBFβ system, that the MET can be reversed, thus demonstrating the plasticity of CBFβ-mediated EMT. Moreover, the MET can be reversed by expression of the EMT transcription factor Slug whose expression is dependent on CBFβ. Finally, we demonstrate that loss of CBFβ inhibits the ability of metastatic breast cancer cells to invade bone cell cultures and suppresses their ability to form bone metastases in vivo. Together our findings demonstrate that CBFβ can determine the plasticity of the metastatic cancer cell phenotype, suggesting that its regulation in different micro-environments may play a key role in the establishment of metastatic tumours.
BackgroundHypoxia stimulates metastasis in cancer and is linked to poor patient prognosis. In tumours, oxygen levels vary and hypoxic regions exist within a generally well-oxygenated tumour. However, whilst the heterogeneous environment is known to contribute to metastatic progression, little is known about the mechanism by which heterogeneic hypoxia contributes to cancer progression. This is largely because existing experimental models do not recapitulate the heterogeneous nature of hypoxia.The primary effector of the hypoxic response is the transcription factor Hypoxia inducible factor 1-alpha (HIF1-alpha). HIF1-alpha is stabilised in response to low oxygen levels in the cellular environment and its expression is seen in hypoxic regions throughout the tumour.MethodsWe have developed a model system in which HIF1-alpha can be induced within a sub-population of cancer cells, thus enabling us to mimic the effects of heterogeneic HIF1-alpha expression.ResultsWe show that induction of HIF1-alpha not only recapitulates elements of the hypoxic response in the induced cells but also results in significant changes in proliferation, gene expression and mammosphere formation within the HIF1-alpha negative population.ConclusionsThese findings suggest that the HIF1-alpha expressing cells found within hypoxic regions are likely to contribute to the subsequent progression of a tumour by modifying the behaviour of cells in the non-hypoxic regions of the local micro-environment.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4577-1) contains supplementary material, which is available to authorized users.
General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms Eukaryotic transcription initiation by RNA polymerase II requires numerous transcription factors and cofactors to nucleate at core promoter to form a pre-initiation complex (1, 2). The core promoter plays a critical role during transcriptional initiation and contains a number of DNA sequence elements such as the TATA box, the initiator, the downstream promoter element, the TFIIB recognition elements (BREs) and others (1-4). These elements are recognized by general transcription factors and cofactors (5-9), and assist to direct and orientate pre-initiation complex formation at the promoter. Core promoter elements not only regulate the activity of transcription but also determine transcription start site selection (4, 10 and 11). Genome-wide studies have revealed that many genes lack so-called 'canonical' core promoter elements, suggesting that other core promoter elements remain to be discovered. Indeed, a recent study showed that core promoter element, DTIE, directs transcription start site selection of genes with TATA-less promoters (12). Nevertheless, it has been proposed that core promoter elements may not be essential to transcription of some genes in vivo (13), suggesting that canonical core promoter elements fine-tune physiological responses for specific genes (4). It has been shown that many 'noncanonical' promoters instead contain epigenetic marks including histone modifications (H3K4me3 and H3K27me3) and DNA marks such as enhancers, CpG islands and ATG deserts (14-16). The mechanisms by which canonical or noncanonical core promoters regulate transcriptional initiation is not fully understood.Transcription factor TFIIA comprises of three subunits, α, β and γ; TFIIAα/β and TFIIAγ are encoded by different genes (17-19). The precursor of TFIIAα/β can be digested by taspase1, but uncleaved TFIIAα/β remains active in transcriptional regulation (20). Recent studies showed that the cleavage of TFIIA by taspase 1 is involved in a number of molecular and biological processes (21-24). Although TFIIA was originally characterized as a general transcription factor, TFIIA is dispensable in transcription in vitro (25-26); perhaps, TFIIA is better to be described as a general cofactor because it acts as an anti-repressor or coactivator in transcriptional regulation (27)(28)(29)(30)(31). TFIIA can counteract the inhibitory roles of TAF1 and BTAF1 during TBP binding to the TATA box as well as the repressive effects of NC2 and HMGB1 on transcription (27, 28). TFIIA has also been shown to stabilize TFIID binding to DNA by interacting with transcriptional activators, TBP-associated factors (29, 30,(32)(33)(34) and TBP-related factors (35-37). It has been proposed that TFIIA induces the disassociation of TBP dimers and promotes the association between TBP and the TATA-box promoter (38). TFIIA stabil...
Core binding factor b (CBFb), the essential coregulator of RUNX transcription factors, is one of the most frequently mutated genes in estrogen receptor-positive (ER þ) breast cancer. Many of these mutations are nonsense mutations and are predicted to result in loss of function, suggesting a tumor suppressor role for CBFb. However, the impact of missense mutations and the loss of CBFb in ER þ breast cancer cells have not been determined. Here we demonstrate that missense mutations in CBFb accumulate near the Runt domain-binding region. These mutations inhibit the ability of CBFb to form CBFb-Runx-DNA complexes. We further show that deletion of CBFb, using CRISPR-Cas9, in ER þ MCF7 cells results in an increase in cell migration. This increase in migration is dependent on the presence of ERa. Analysis of the potential mechanism revealed that the increase in migration is driven by the coregulation of Trefoil factor 1 (TFF1) by CBFb and ERa. RUNX1-CBFb acts to repress ERa-activated expression of TFF1. TFF1 is a motogen that stimulates migration and we show that knockdown of TFF1 in CBFb À/À cells inhibits the migratory phenotype. Our findings reveal a new mechanism by which RUNX1-CBFb and ERa combine to regulate gene expression and a new role for RUNX1-CBFb in the prevention of cell migration by suppressing the expression of the motogen TFF1. Implications: Mutations in CBFb contribute to the development of breast cancer by inducing a metastatic phenotype that is dependent on ER.
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