Epigenetic events, which are somatically inherited through cell division, are potential drivers of acquired drug resistance in cancer. The high rate of epigenetic change in tumours generates diversity in gene expression patterns that can rapidly evolve through drug selection during treatment, leading to the development of acquired resistance. This will potentially confound stratified chemotherapy decisions that are solely based on mutation biomarkers. Poised epigenetic states in tumour cells may drive multistep epigenetic fixation of gene expression during the acquisition of drug resistance, which has implications for clinical strategies to prevent the emergence of drug resistance.
Chromatin constitutes a repressive barrier to the process of ligand-dependent transcriptional activity of nuclear receptors. Nucleosomes prevent the binding of estrogen receptor ? (ER?) in absence of ligand and thus represent an important level of transcriptional regulation. Here, we show that in breast cancer MCF-7 cells, TLE3, a co-repressor of the Groucho/Grg/TLE family, interacts with FoxA1 and is detected at regulatory elements of ER? target genes in absence of estrogen. As a result, the chromatin is maintained in a basal state of acetylation, thus preventing ligand-independent activation of transcription. In absence of TLE3, the basal expression of ER? target genes induced by E2 is increased. At the TFF1 gene, the recruitment of TLE3 to the chromatin is FoxA1-dependent and prevents ER? and RNA polymerase II recruitment to TFF1 gene regulatory elements. Moreover, the interaction of TLE3 with HDAC2 results in the maintenance of acetylation at a basal level. We also provide evidence that TLE3 is recruited at several other regulatory elements of ER? target genes and is probably an important co-regulator of the E2 signaling pathway. In sum, our results describe a mechanism by which TLE3 affects ligand dependency in ER?-regulated gene expression via its binding restricting function and its role in gene regulation by histone acetylation.
Resistance to anti-estrogen therapies is a major cause of disease relapse and mortality in estrogen receptor alpha (ER?)-positive breast cancers. Tamoxifen or estrogen withdrawal increases the dependence of breast cancer cells on Notch signalling. Here, we investigated the contribution of Nicastrin and Notch signalling in endocrine-resistant breast cancer cells.
Tumor characteristics are decisive in the determination of treatment strategy for patients with breast cancer. Patients with estrogen receptor ? (ER?)-positive breast cancer can benefit from long-term hormonal treatment. Nonetheless, the majority of patients will develop resistance to these therapies. Here, we investigated the role of the nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in antiestrogen-sensitive and -resistant breast cancer cells. We identified genome-wide LRH-1-binding sites using ChIP-seq (chromatin immunoprecipitation sequencing), uncovering preferential binding to regions distal to transcriptional start sites. We further characterized these LRH-1-binding sites by integrating overlapping layers of specific chromatin marks, revealing that many LRH-1-binding sites are active and could be involved in long-range enhancer-promoter looping. Combined with transcriptome analysis of LRH-1-depleted cells, these results show that LRH-1 regulates specific subsets of genes involved in cell proliferation in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Furthermore, the LRH-1 transcriptional program is highly associated with a signature of poor outcome and high-grade breast cancer tumors in vivo. Herein, we report the genome-wide location and molecular function of LRH-1 in breast cancer cells and reveal its therapeutic potential for the treatment of breast cancers, notably for tumors resistant to treatments currently used in therapies.
Chronic periaortitis (CP) usually responds to glucocorticoids, but some patients have glucocorticoid-refractory disease or contraindications to glucocorticoid therapy. This study was undertaken to evaluate treatment with the anti-interleukin-6 receptor (anti-IL-6R) antibody tocilizumab in 2 patients with CP, one with refractory disease and the other with contraindications to glucocorticoids, and to assess IL-6 levels in an additional cohort of patients with CP.
The estrogen receptor (ER)? drives growth in two-thirds of all breast cancers. Several targeted therapies, collectively termed endocrine therapy, impinge on estrogen-induced ER? activation to block tumor growth. However, half of ER?-positive breast cancers are tolerant or acquire resistance to endocrine therapy. We demonstrate that genome-wide reprogramming of the chromatin landscape, defined by epigenomic maps for regulatory elements or transcriptional activation and chromatin openness, underlies resistance to endocrine therapy. This annotation reveals endocrine therapy-response specific regulatory networks where NOTCH pathway is overactivated in resistant breast cancer cells, whereas classical ER? signaling is epigenetically disengaged. Blocking NOTCH signaling abrogates growth of resistant breast cancer cells. Its activation state in primary breast tumors is a prognostic factor of resistance in endocrine treated patients. Overall, our work demonstrates that chromatin landscape reprogramming underlies changes in regulatory networks driving endocrine therapy resistance in breast cancer.
The oestrogen receptor alpha (ESR1) is a transcription factor that potentiates the response to diverse stimuli, including oestrogen and growth factors, in various tissue types. Its recruitment to the DNA is directly regulated by the chromatin landscape, inclusive of chromatin compaction and epigenetic modifications. In this review we discuss our current understanding of the interplay between ESR1 signaling and the chromatin landscape. We present how the chromatin landscape primes the lineage-specific response and contributes to stimuli-specific signaling. Finally, we discuss recent efforts to decipher the relationship between genetic and epigenetic as it relates to ESR1 signaling in breast cancer.
Treatment of large-vessel vasculitis (LVV) remains challenging. Patients usually respond to glucocorticoid (GC) therapy, but often relapse on tapering of the GC dose or after GC withdrawal. In addition, GCs are fraught with numerous adverse events. The aim of this study was to assess the efficacy and safety of the anti-IL-6 receptor (IL-6R) antibody tocilizumab (TCZ) in patients with LVV.
Biological agents have originally been developed to treat refractory arthritis, but evidence has been accruing, supporting their use in vasculitis as well. In the large-vessel vasculitides giant cell arteritis and Takayasu arteritis, TNF-? inhibitors have shown some efficacy in patients with relapsing disease. In contrast, in patients with recent onset of giant cell arteritis, TNF-? inhibitors failed to provide a significant benefit over and above that conferred by glucocorticoids alone. More recent, preliminary data suggest a role for the interleukin-6 receptor antagonist tocilizumab in both resistant and treatment-naïve giant cell arteritis and Takayasu arteritis. Biological agents have also been proposed to treat difficult anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis. Uncontrolled observations suggest that the TNF-? inhibitor infliximab might be beneficial in resistant cases. On the contrary, a randomized controlled trial did not show superiority of the recombinant human soluble TNF-? p75 receptor fusion protein etanercept over placebo in maintaining remission in granulomatosis with polyangiitis. Two randomized controlled trials have demonstrated that the anti-CD20 monoclonal antibody rituximab was as effective as the standard-of-care agent cyclophosphamide in inducing remission. In addition, rituximab appeared to be superior to cyclophosphamide in inducing remission in the subset of patients with relapsing disease. These findings prove that biological therapy has a role in vasculitis. Research is investigating novel therapies as well as focusing on how to best use the available drugs.
Chromatin is a well-known obstacle to transcription as it controls DNA accessibility, which directly impacts the recruitment of the transcriptional machinery. The recent burst of functional genomic studies provides new clues as to how transcriptional competency is regulated in this context. In this review, we discuss how these studies have shed light on a specialized subset of transcription factors, defined as pioneer factors, which direct recruitment of downstream transcription factors to establish lineage-specific transcriptional programs. In particular, we present evidence of an interplay between pioneer factors and the epigenome that could be central to this process. Finally, we discuss how pioneer factors, whose expression and function are altered in tumors, are also being considered for their prognostic value and should therefore be regarded as potential therapeutic targets. Thus, pioneer factors emerge as key players that connect the epigenome and transcription in health and disease.
Altered transcriptional programs are a hallmark of diseases, yet how these are established is still ill-defined. PBX1 is a TALE homeodomain protein involved in the development of different types of cancers. The estrogen receptor alpha (ER?) is central to the development of two-thirds of all breast cancers. Here we demonstrate that PBX1 acts as a pioneer factor and is essential for the ER?-mediated transcriptional response driving aggressive tumors in breast cancer. Indeed, PBX1 expression correlates with ER? in primary breast tumors, and breast cancer cells depleted of PBX1 no longer proliferate following estrogen stimulation. Profiling PBX1 recruitment and chromatin accessibility across the genome of breast cancer cells through ChIP-seq and FAIRE-seq reveals that PBX1 is loaded and promotes chromatin openness at specific genomic locations through its capacity to read specific epigenetic signatures. Accordingly, PBX1 guides ER? recruitment to a specific subset of sites. Expression profiling studies demonstrate that PBX1 controls over 70% of the estrogen response. More importantly, the PBX1-dependent transcriptional program is associated with poor-outcome in breast cancer patients. Correspondingly, PBX1 expression alone can discriminate a priori the outcome in ER?-positive breast cancer patients. These features are markedly different from the previously characterized ER?-associated pioneer factor FoxA1. Indeed, PBX1 is the only pioneer factor identified to date that discriminates outcome such as metastasis in ER?-positive breast cancer patients. Together our results reveal that PBX1 is a novel pioneer factor defining aggressive ER?-positive breast tumors, as it guides ER? genomic activity to unique genomic regions promoting a transcriptional program favorable to breast cancer progression.
Methylation of the lysine 9 residue of histone H3 (H3K9) is linked to transcriptional repression. The observed structure of chromatin in porcine and murine embryos is different with regard to H3K9 dimethylation status, leading to our hypothesis that the intracellular mechanisms responsible for H3K9 methylation would also differ between these two species. The objectives of this study were: (1) to determine the extent that DNA, mRNA, and protein synthesis serve in maintaining the asymmetrical distribution of dimethylated H3K9 in porcine zygotes, (2) determine the extent to which the intracellular localization of individual pronuclei correlated with H3K9 dimethylation status, and (3) to determine the abundance of transcripts encoding the histone methyltransferases, with H3K9 methylation activity, in porcine oocytes and embryos. Our findings are that (1) H3K9 dimethylation status is not affected by DNA replication, transcription, or protein synthesis, (2) the location of a pronucleus does not significantly affect the H3K9 dimethylation status of the chromatin within that pronucleus, and (3) the histone methyltransferases with activity for H3K9 differ in transcript abundance in porcine oocytes and cleavage stage embyros. These results support our hypothesis that there is a difference in intracellular mechanisms affecting dimethylation status of H3K9 between porcine and murine embryos.
During blastocyst formation the segregation of the inner cell mass (ICM) and trophectoderm is governed by the mutually antagonistic effects of the transcription factors Oct4 and Cdx2. Evidence indicates that suppression of Oct4 expression in the trophectoderm is mediated by Cdx2. Nonetheless, the underlying epigenetic modifiers required for Cdx2-dependent repression of Oct4 are largely unknown. Here we show that the chromatin remodeling protein Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. By employing a combination of RNA interference (RNAi) and gene expression analysis we found that both Brg1 Knockdown (KD) and Cdx2 KD blastocysts exhibit widespread expression of Oct4 in the trophectoderm. Interestingly, in Brg1 KD blastocysts and Cdx2 KD blastocysts, the expression of Cdx2 and Brg1 is unchanged, respectively. To address whether Brg1 cooperates with Cdx2 to repress Oct4 transcription in the developing trophectoderm, we utilized preimplantation embryos, trophoblast stem (TS) cells and Cdx2-inducible embryonic stem (ES) cells as model systems. We found that: (1) combined knockdown (KD) of Brg1 and Cdx2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of Brg1 or Cdx2 alone, (2) endogenous Brg1 co-immunoprecipitated with Cdx2 in TS cell extracts, (3) in blastocysts Brg1 and Cdx2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells Brg1 and Cdx2 are recruited to the Oct4 promoter. Lastly, to determine how Brg1 may induce epigenetic silencing of the Oct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of Brg1 KD blastocysts. This analysis revealed that Brg1-dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. In toto, these results demonstrate that Brg1 cooperates with Cdx2 to repress Oct4 expression in the developing trophectoderm to ensure normal development.
During nuclear transfer, reprogramming makes the donor nucleus capable of directing development of the reconstructed embryo. In most cases reprogramming is incomplete, which leads to abnormal expression of early embryonic genes and subsequently, to reduced developmental potential. In the present study, we monitored the expression of Oct4, Nanog, and Sox2 in cloned porcine embryos and evaluated whether serial nuclear transfer, the transfer of nuclei of cloned embryos into enucleated oocytes, has the potential to provide a more complete reprogramming of the donor genome. The data suggested that Nanog and Sox2 expression is properly reactivated after nuclear transfer, but the relative abundance of Oct4 transcripts is abnormally low in cloned porcine blastocysts compared to control embryos produced by in vitro fertilization. When the nuclei of 8- to 16-cell stage cloned embryos were introduced into enucleated oocytes to expose the chromosomes repeatedly to the ooplasmic factors, the resulting embryos showed poor developmental potential: a significantly lower percentage of embryos developed to the 4-cell (12.0% vs. 31.8%), 8-cell (3.1% vs. 15.0%) and blastocyst (0% vs. 8.7%) stages compared to those produced following a single round of nuclear transfer (P < 0.05). The additional time for reprogramming also did not improve gene expression. By the late 4-cell stage, Oct4 and Sox2 expression levels were low in serial nuclear transfer embryos compared to those in embryos generated by in vitro fertilization or nuclear transfer. Overall, both developmental and gene expression data indicated that reprogramming of the donor nucleus could not be improved by serial nuclear transfer in the pig.
Histone methylation plays an important role in regulating chromatin structure and gene expression. Methylation of the lysine residue 27 of histone H3 (H3K27) is an epigenetic mark that is closely linked with transcriptional repression; global patterns of H3K27 methylation undergo dramatic changes during cleavage development in the mouse. The aim of this study was to characterize the H3K27 methylation pattern in cleavage stage porcine embryos obtained either by in vivo or in vitro fertilization or parthenogenetic activation and to determine the expression patterns of EED, EZH2, and SUZ12 (regulators of H3K27 methylation). We found that monomethylated H3K27 was detectable in the nuclei of oocytes and pronuclear, 2-cell, 4-cell, 8-cell, and blastocyst stage embryos. Trimethylated H3K27 was detectable in the nuclei of GV stage oocytes, the chromosome of MII stage oocytes and a single pronucleus of the pronuclear stage embryos produced by fertilization; the signals were faint or absent in nuclei of two-cell through blastocyst stage embryos. In addition, EED transcripts were increased from the four-cell stage (P < 0.05) in embryos obtained by in vitro fertilization, parthenogenetic activation and in vivo fertilization. EZH2 transcript levels were highest in the GV-stage oocyte (P < 0.05). SUZ12 transcripts were transiently increased at the four-cell stage (P < 0.05) in parthenogenetic and in vivo derived embryos. Our results suggest that H3K27 trimethylation is an epigenetic marker of maternally derived chromatin that is globally remodeled during porcine embryogenesis.
Epigenetic reprogramming plays a pivotal role during embryogenesis, including both covalent and non-covalent modifications to chromatin. In this study, we investigated the role of SNF2 chromatin remodeling ATPases (SMARCA2 (previously known as BRAHMA), SMARCA4 (previously known as BRG1), SMARCA5 (previously known as SNF2H), SMARCA1 (previously known as SNF2L), CHD3, and CHD5) during porcine preimplantation embryonic development. Transcript levels for these ATPases change dynamically throughout development. We also investigated the effect of altering transcript levels of SMARCA2 and SMARCA4 via mRNA injection. Overexpression of SMARCA2 and SMARCA4 severely impaired embryo development. Results from these experiments show that embryos injected with SMARCA2 mRNA arrest between the four-cell and blastocyst stages. However, embryos injected with either wild-type SMARCA4 or a dominant negative variant or SMARCA4 arrest before zygotic genome activation. No differences in transcript abundance of SOX2, POU5F1, NANOG, and EIF1 (previously known as eIF1A) were detected after injection with SMARCA2 or its dominant negative variant at 48 h post-injection. Conversely, embryos injected with wild-type SMARCA4 and its dominant negative variant possessed altered expression of these genes. Examination of SNF2-type ATPase transcript abundance across all treatment groups revealed that only SMARCA1 was altered following injection with wild-type SMARCA2 and wild-type and dominant negative SMARCA4. We conclude that the arrest in porcine embryo development observed after injection is specific to the ATPase injected. Our data strongly support the hypothesis that SMARCA2 and SMARCA4 play different but fundamental roles controlling gene expression during early mammalian embryogenesis.
Over two-thirds of breast cancers rely on estrogen receptor ? (ER?) for their growth. Endocrine therapies antagonize estrogen-dependent ER? activation but resistance to these treatments occurs and is associated with poor prognosis. Crosstalk between alternative survival pathways and ER? are currently held as the primary cause of resistance. However, blocking these pathways does not cure endocrine therapy resistant breast cancer suggesting the existence of additional mechanisms. While cancer is commonly considered a genetic disease, the importance of epigenetic events in promoting tumor initiation and progression is increasingly recognized. Here, we consider how epigenetic modifications and alterations to the chromatin landscape contribute to endocrine therapy resistance by modulating ER? expression or altering its genomic activity.
Glucocorticoids are the mainstay of treatment of idiopathic retroperitoneal fibrosis (IRF). However, relapses are frequent upon tapering of the glucocorticoid dose. A variety of traditional immunosuppressants have been proposed as steroid-sparing agents, but some patients fail to adequately respond to combined glucocorticoid and immunosuppressive therapy. We report a patient with IRF refractory to combined glucocorticoid and methotrexate therapy treated with the anti-TNF-? monoclonal antibody infliximab. Infliximab was administered at 5 mg/kg/bodyweight at week 0, 2, 6 and 8-weekly thereafter for 3 consecutive years. Drug efficacy and safety were assessed clinically and by laboratory tests at treatment onset and subsequently before each infusion. In addition, 18FFluorodeoxyglucose (FDG) positron emission computerised tomography (PET/CT) and abdominal CT scans were used to monitor disease activity and response to treatment. Infliximab therapy resulted in a satisfactory clinical and laboratory response paralleled by an improvement in imaging findings. No serious adverse events were noted. Infliximab may be an effective and safe treatment for refractory IRF. A controlled study is required to confirm our findings.
PBX1 is a TALE homeodomain transcription factor involved in organogenesis and tumorigenesis. Although it has been shown that ovarian, breast, and melanoma cancer cells depend on PBX1 for cell growth and survival, the molecular mechanism of how PBX1 promotes tumorigenesis remains unclear. Here, we applied an integrated approach by overlapping PBX1 ChIP-chip targets with the PBX1-regulated transcriptome in ovarian cancer cells to identify genes whose transcription was directly regulated by PBX1. We further determined if PBX1 target genes identified in ovarian cancer cells were co-overexpressed with PBX1 in carcinoma tissues. By analyzing TCGA gene expression microarray datasets from ovarian serous carcinomas, we found co-upregulation of PBX1 and a significant number of its direct target genes. Among the PBX1 target genes, a homeodomain protein MEOX1 whose DNA binding motif was enriched in PBX1-immunoprecipicated DNA sequences was selected for functional analysis. We demonstrated that MEOX1 protein interacts with PBX1 protein and inhibition of MEOX1 yields a similar growth inhibitory phenotype as PBX1 suppression. Furthermore, ectopically expressed MEOX1 functionally rescued the PBX1-withdrawn effect, suggesting MEOX1 mediates the cellular growth signal of PBX1. These results demonstrate that MEOX1 is a critical target gene and cofactor of PBX1 in ovarian cancers.
Anti-TNF-? therapy has successfully been used to treat Takayasu arteritis (TA) refractory to conventional immunosuppressive treatment. However, some patients fail to respond even to TNF-? blockers. Interleukin-6 (IL-6) is a key player in the pathogenesis of TA. Preliminary data also suggest efficacy of the IL-6 receptor inhibitor tocilizumab in patients with large-vessel vasculitis. We report a patient with TA refractory to multiple conventional immunosuppressive agents and two TNF-? blockers successfully treated with monthly tocilizumab infusions (8 mg/kg body weight) for 6 consecutive months. Clinical indices of disease activity, inflammatory markers, and 18Ffluorodeoxyglucose positron emission/computerised tomography findings normalised, while the prednisone dosage could be tapered. Serum IL-6 and soluble IL-6 receptor (sIL-6R) levels raised during tocilizumab treatment consistent with the mode of action of tocilizumab. Tocilizumab holds promise for patients with refractory TA. Larger studies are required to confirm our findings.
Coordinated partitioning of intracellular cargoes between nuclear and cytoplasmic compartments is critical for cell survival and differentiation. The karyopherin ?/? heterodimer functions to import cytoplasmic proteins that possess classical nuclear localisation signals into the nucleus. Seven karyopherin?subtypes have been identified in mammals. The aim of this study was to determine the relative abundance of transcripts encoding seven karyopherin?subtypes in porcine oocytes and embryos at discrete stages of cleavage development, and to determine the developmental requirements of karypopherin?7 (KPNA7), an oocyte and cleavage stage embryo-specific karyopherin?subtype. We hypothesised that knockdown of KPNA7 would negatively affect porcine cleavage development. To test this hypothesis, in vitro matured and fertilised porcine oocytes were injected with a double-stranded interfering RNA molecule that targeted KPNA7; nuclei were counted in all embryos 6 days after fertilisation. Embryos injected with KPNA7-interfering RNAs possessed significantly lower cell numbers than their respective control groups (P<0.05). In vitro binding assays also suggest that KPNA7 may transport intracellular proteins that possess unique nuclear localisation signals. Our data suggest that embryos have differential requirements for individual karyopherin?subtypes and that these karyopherin?subtypes differentially transport intracellular cargo during cleavage development.
The nuclear receptor family includes 48 transcription factors with fundamental and diverse roles in development, metabolism and disease. Differently from other transcription factors, they engage with well-defined DNA regulatory elements mostly after ligand induced structural changes. However, nuclear receptor binding is not stochastic and only a fraction of regulatory elements actively engages with these factors. In this review we summarize recent advances in the understanding of the interaction between nuclear receptors and the DNA. We discuss how chromatin accessibility and epigenetic modifications contribute to recruitment and transactivation of nuclear receptors. Lastly, we present novel evidence of the interplay between non-coding RNA nuclear receptors in mediating the assembly of the transcriptional machinery.
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