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The field of stem cell-based embryo-like models is rapidly evolving, providing in vitro models of in utero stages of mammalian development. Here, we detail steps to first establish adherent spheroids composed of three cell types from mouse embryonic stem cells solely treated with a chemical inhibitor of SUMOylation. We then describe procedures for generating highly reproducible gastruloids from these dissociated spheroid cells, as well as embryo-like structures comprising anterior neural and trunk somite-like regions using an optimized microfluidics platform. For complete details on the use and execution of this protocol, please refer to Cossec et al. (2023). 1 ., Competing Interests: Declaration of interests Authors are designated as inventors of the patent application WO/2023/002057 covering aspects of the in vitro generation of organized 3D cell structures and the microfluidic chip described in the article., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent advances in synthetic embryology have opened new avenues for understanding the complex events controlling mammalian peri-implantation development. Here, we show that mouse embryonic stem cells (ESCs) solely exposed to chemical inhibition of SUMOylation generate embryo-like structures comprising anterior neural and trunk-associated regions. HypoSUMOylation-instructed ESCs give rise to spheroids that self-organize into gastrulating structures containing cell types spatially and functionally related to embryonic and extraembryonic compartments. Alternatively, spheroids cultured in a droplet microfluidic device form elongated structures that undergo axial organization reminiscent of natural embryo morphogenesis. Single-cell transcriptomics reveals various cellular lineages, including properly positioned anterior neuronal cell types and paraxial mesoderm segmented into somite-like structures. Transient SUMOylation suppression gradually increases DNA methylation genome wide and repressive mark deposition at Nanog. Interestingly, cell-to-cell variations in SUMOylation levels occur during early embryogenesis. Our approach provides a proof of principle for potentially powerful strategies to explore early embryogenesis by targeting chromatin roadblocks of cell fate change., Competing Interests: Declaration of interests J.-C.C., T.T., S.S., C.N.B., and A.D. are designated as inventors of the patent application WO 2023/002057 A2 covering the aspects of the in vitro generation of organized 3D cell structures and the microfluidic device described in the manuscript., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Antiestrogens (AEs) are widely used for treatment of estrogen receptor alpha (ERα)-positive breast cancer, but display variable degrees of partial agonism in estrogen target tissues and breast cancer (BC) cells. The fact that BC cells resistant to selective ER modulators (SERMs) like tamoxifen (Tam) can still be sensitive to pure AEs, also called selective ER downregulators, suggests different mechanisms of action, some of which may contribute to the more complete suppression of estrogen target genes by pure AEs. We report herein that pure AEs such as fulvestrant induce transient binding of ERα to DNA, followed by rapid release after 30-40 min without loss of nuclear localization. Loss of DNA binding preceded receptor degradation and was not prevented by proteasome inhibition. Chromatin was less accessible in the presence of fulvestrant than with estradiol or Tam as early as 20 min following treatment, suggesting that chromatin remodeling by pure AEs at ERα target regions prevents transcription in spite of receptor binding. SUMO2/3 marks were detected on chromatin at the peak of ERα binding in cells treated with pure AEs, but not SERMs. Furthermore, decreasing SUMOylation by overexpressing the deSUMOylase SENP1 significantly delayed receptor release from DNA and de-repressed expression of estrogen target genes in the presence of fulvestrant, both in ERα-expressing MCF-7 cells and in transiently transfected ER-negative SK-BR-3 cells. Finally, mutation V534E, identified in a breast metastasis resistant to hormonal therapies, prevented ERα modification and resulted in increased transcriptional activity of estrogen target genes in the presence of fulvestrant in SK-BR-3 cells. Together, our results establish a role for SUMOylation in achieving a more complete transcriptional shut-off of estrogen target genes by pure AEs vs. SERMs in BC cells.

Enhancers are intergenic DNA elements that regulate the transcription of target genes in response to signaling pathways by interacting with promoters over large genomic distances. Recent studies have revealed that enhancers are bi-directionally transcribed into enhancer RNAs (eRNAs). Using single-molecule fluorescence in situ hybridization (smFISH), we investigated the eRNA-mediated regulation of transcription during estrogen induction in MCF-7 cells. We demonstrate that eRNAs are localized exclusively in the nucleus and are induced with similar kinetics as target mRNAs. However, eRNAs are mostly nascent at enhancers and their steady-state levels remain lower than those of their cognate mRNAs. Surprisingly, at the single-allele level, eRNAs are rarely co-expressed with their target loci, demonstrating that active gene transcription does not require the continuous transcription of eRNAs or their accumulation at enhancers. When co-expressed, sub-diffraction distance measurements between nascent mRNA and eRNA signals reveal that co-transcription of eRNAs and mRNAs rarely occurs within closed enhancer-promoter loops. Lastly, basal eRNA transcription at enhancers, but not E2-induced transcription, is maintained upon depletion of MLL1 and ERα, suggesting some degree of chromatin accessibility prior to signal-dependent activation of transcription. Together, our findings suggest that eRNA accumulation at enhancer-promoter loops is not required to sustain target gene transcription., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Purpose of Review: Update on the most recent clinical evidence on selective estrogen receptor degraders (SERDs) in the treatment of hormone receptor (HR)-positive (HR +), human epidermal growth factor receptor 2 (HER2)-negative (HER2-) breast cancer. Recent Findings: Despite effective endocrine therapies, resistance commonly develops during treatment of HR + breast cancer and mutations in ESR1 account for a large proportion of resistance mechanisms. After demonstration of the superior efficacy of fulvestrant in ESR1-mutated tumors, recent advances allowed the development of a novel class of orally bioavailable selective estrogen receptor degraders (SERDs), which are beginning to revolutionize the field. The first approved oral SERD, elacestrant, is currently used in the second-line treatment of HR + /HER2- metastatic breast cancer, and a number of other oral SERDs are undergoing clinical evaluation in both the metastatic and early-stage settings. Summary: SERDs are a rapidly developing class of antiestrogens that show activity in treatment of tumors harboring ESR1 mutations associated with resistance to earlier generations of endocrine therapy, but knowledge gaps remain, and further research is necessary to better define their optimal use. [ABSTRACT FROM AUTHOR]

Post-translational protein modifications (PTMs) significantly enhance the functional diversity of proteins and are therefore important for the expansion and the dynamics of the cell's proteome. In addition to structurally simpler PTMs, substrates also undergo modification through the reversible attachment of small proteins. The best understood PTM of this nature to date is the covalent conjugation of ubiquitin and ubiquitin-like proteins (UBLs) to their substrates. The protein family of small ubiquitin-like modifier (SUMO) is one of these UBLs that has received increasing scientific attention. The pathway of SUMOylation is highly conserved in all eukaryotic cells and is crucial for their survival. It plays an essential role in many biological processes, such as the maintenance of genomic integrity, transcriptional regulation, gene expression, and the regulation of intracellular signal transduction, and thereby influences DNA damage repair, immune responses, cell cycle progression, and apoptosis. Several studies have already shown that in this context protein SUMOylation is involved in the control mechanisms of various cellular receptors. This article unites data from different studies focusing on the investigation of the strictly conserved three-step enzyme cascade of protein SUMOylation and the functional analysis of the involved proteins E1, E2, and E3 and SUMOylation target proteins. Furthermore, this review highlights the role of nuclear receptor SUMOylation and its importance for the cellular functionality and disease development arising from defects in correct protein SUMOylation. [ABSTRACT FROM AUTHOR]

Proteins are the most common types of biomarkers used in breast cancer (BC) theranostics and management. By definition, a biomarker must be a relevant, objective, stable, and quantifiable biomolecule or other parameter, but proteins are known to exhibit the most variate and profound structural and functional variation. Thus, the proteome is highly dynamic and permanently reshaped and readapted, according to changing microenvironments, to maintain the local cell and tissue homeostasis. It is known that protein posttranslational modifications (PTMs) can affect all aspects of protein function. In this review, we focused our analysis on the different types of PTMs of histological biomarkers in BC. Thus, we analyzed the most common PTMs, including phosphorylation, acetylation, methylation, ubiquitination, SUMOylation, neddylation, palmitoylation, myristoylation, and glycosylation/sialylation/fucosylation of transcription factors, proliferation marker Ki-67, plasma membrane proteins, and histone modifications. Most of these PTMs occur in the presence of cellular stress. We emphasized that these PTMs interfere with these biomarkers maintenance, turnover and lifespan, nuclear or subcellular localization, structure and function, stabilization or inactivation, initiation or silencing of genomic and non-genomic pathways, including transcriptional activities or signaling pathways, mitosis, proteostasis, cell–cell and cell–extracellular matrix (ECM) interactions, membrane trafficking, and PPIs. Moreover, PTMs of these biomarkers orchestrate all hallmark pathways that are dysregulated in BC, playing both pro- and/or antitumoral and context-specific roles in DNA damage, repair and genomic stability, inactivation/activation of tumor-suppressor genes and oncogenes, phenotypic plasticity, epigenetic regulation of gene expression and non-mutational reprogramming, proliferative signaling, endocytosis, cell death, dysregulated TME, invasion and metastasis, including epithelial–mesenchymal/mesenchymal–epithelial transition (EMT/MET), and resistance to therapy or reversal of multidrug therapy resistance. PTMs occur in the nucleus but also at the plasma membrane and cytoplasmic level and induce biomarker translocation with opposite effects. Analysis of protein PTMs allows for the discovery and validation of new biomarkers in BC, mainly for early diagnosis, like extracellular vesicle glycosylation, which may be considered as a potential source of circulating cancer biomarkers. [ABSTRACT FROM AUTHOR]

The evolution of breast cancers results from the emergence of epithelial cell subpopulations containing variant Estrogen Receptor α which is able to bypass conventional treatments aimed at antagonizing the activity of this tumor-promoting receptor. The present investigation concerns a few estradiol derivates bearing substituents in position 11β that might not only contribute to the development of drugs to alleviate this unfortunate issue but that may be also helpful in identifying molecular aspects of resistance to this receptor in order to elaborate other therapeutic approaches. In this regard, AP-1 assisted and ERE-directed ERα transcriptions are demonstrated to be key factors in this area: AP-1 transcriptions are shown to antagonize ERE transcriptions, thereby limiting their tumor-promoting activity. This property results from a conformal change in the receptor, which is induced essentially by estrogenic ligands which, inserted into a cavity of ERα's ligand-binding pocket, govern this regulatory mechanism. Flexible 11β side-chains favor this insertion, in contrast to their rigid counterparts, which counteract it; these properties give rise to strong estrogenic, SERM or SERD profiles. Suspected extracellular regulatory mechanisms resulting from these ligand-induced transcriptions are elaborated on in the present work in the context of breast cancer development. [ABSTRACT FROM AUTHOR]

Immune checkpoint inhibitors (ICIs) show promise as second‐line treatment for advanced bladder cancer (BLCA); however, their responsiveness is limited by the immune evasion mechanisms in tumor cells. This study conduct a Cox regression analysis to screen mRNA‐binding proteins and reveals an association between Ras GTPase‐activating protein‐binding protein 1 (G3BP1) and diminished effectiveness of ICI therapy in patients with advanced BLCA. Subsequent investigation demonstrates that G3BP1 enhances immune evasion in BLCA cells by downregulating major histocompatibility complex class I (MHC‐I) through phosphoinositide 3‐kinase (PI3K)/Akt signaling activation. Mechanistically, G3BP1 interacts with splicing factor synergistic lethal with U5 snRNA 7 (SLU7) to form a complex with poly(A)‐binding protein cytoplasmic 1 and eukaryotic translation initiation factor 4 gamma 1. This complex stabilizes the closed‐loop structure of the mRNAs of class IA PI3Ks and consequently facilitates their translation and stabilization, thereby activating PI3K/Akt signaling to downregulate MHC‐I. Consistently, targeting G3BP1 with epigallocatechin gallate (EGCG) impedes immune evasion and sensitizes BLCA cells to anti‐programmed cell death (PD)‐1 antibodies in mice. Thus, G3BP1 and SLU7 collaboratively contribute to immune evasion in BLCA, indicating that EGCG is a precision therapeutic agent to enhance the effectiveness of anti‐PD‐1 therapy. [ABSTRACT FROM AUTHOR]

Simple Summary: Breast cancer is a common type of cancer among women. One type, estrogen receptor-positive (ER+), is treated with endocrine therapies. However, some patients develop resistance to these therapies, which is a challenge. Scientists have developed second-generation drugs called selective estrogen receptor degraders (SERDs) that can overcome the limitations of the existing treatment. These drugs are taken orally, which is more convenient for patients. SERDS are important because they offer a more effective and less invasive treatment option for patients with ER+ breast cancer who develop resistance to endocrine therapies. Several oral SERDs are currently in clinical trials, which means they are being tested on patients. If they are proven effective, they could become a standard treatment for ER+ breast cancer in the future. Breast cancer is the most common cancer among women worldwide, and estrogen receptor-positive (ER+) breast cancer accounts for a significant proportion of cases. While various treatments are available, endocrine therapies are often the first-line treatment for this type of breast cancer. However, the development of drug resistance poses a significant challenge in managing this disease. ESR1 mutations have been identified as a common mechanism of endocrine therapy resistance in ER+ breast cancer. The first-generation selective estrogen receptor degrader (SERD) fulvestrant has shown some activity against ESR1 mutant tumors. However, due to its poor bioavailability and need for intramuscular injection, it may not be the optimal therapy for patients. Second-generation SERDs were developed to overcome these limitations. These newer drugs have improved oral bioavailability and pharmacokinetics, making them more convenient and effective for patients. Several oral SERDs are now in phase III trials for early and advanced ER+ breast cancer. This review summarizes the background of oral SERD development, the current status, and future perspectives. [ABSTRACT FROM AUTHOR]

The antisense RNA molecule is a unique DNA transcript consisting of 19-23 nucleotides, characterized by its complementary nature to mRNA. These antisense RNAs play a crucial role in regulating gene expression at various stages, including replication, transcription, and translation. Additionally, artificial antisense RNAs have demonstrated their ability to effectively modulate gene expression in host cells.Consequently, there has been a substantial increase in research dedicated to investigating the roles of antisense RNAs. These molecules have been found to be influential in various cellular processes, such as X-chromosome inactivation and imprinted silencing in healthy cells. However, it is important to recognize that in cancer cells; aberrantly expressed antisense RNAs can trigger the epigenetic silencing of tumor suppressor genes. Moreover, the presence of deletion-induced aberrant antisense RNAs can lead to the development of diseases through epigenetic silencing. One area of drug development worth mentioning is antisense oligonucleotides (ASOs), and a prime example of an oncogenic trans-acting long noncoding RNA (lncRNA) is HOTAIR (HOX transcript antisense RNA). NATs (noncoding antisense transcripts) are dysregulated in many cancers, and researchers are just beginning to unravel their roles as crucial regulators of cancer's hallmarks, as well as their potential for cancer therapy. In this review, we summarize the emerging roles and mechanisms of antisense RNA and explore their application in cancer therapy. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and is typically treated with the FOLFOX regimen (folinic acid, 5‐fluorouracil, and oxaliplatin). However, oxaliplatin resistance remains a serious clinical problem. In the present study, we found that SUMO2/3 was overexpressed in CRC tissues and exogenous overexpression of SUMO2/3 promoted CRC cell proliferation, extension, and invasion and positively regulated the cell cycle. In contrast, SUMO2/3 gene knockdowns inhibited migration and repressed cell viability in vitro and in vivo. In addition, we found that SUMO2/3 was recruited to the cell nucleus and suppressed oxaliplatin‐induced apoptosis of CRC cells. Moreover, Ku80, a DNA‐binding protein essential for the repair of DNA double‐strand breaks, was confirmed to bind with SUMO2/3. Notably, Ku80 undergoes SUMOylation at K307 by SUMO2/3 and this correlated with apoptosis in CRC cells suffering oxaliplatin stress. Collectively, we found that SUMO2/3 plays a specific role in CRC tumorigenesis and acts through Ku80 SUMOylation which is linked with the development of CRC‐oxaliplatin resistance. [ABSTRACT FROM AUTHOR]

During early mammalian embryonic development, fertilized one-cell embryos develop into pre-implantation blastocysts and subsequently establish three germ layers through gastrulation during post-implantation development. In recent years, stem cells have emerged as a powerful tool to study embryogenesis and gastrulation without the need for eggs, allowing for the generation of embryo-like structures known as synthetic embryos or embryoids. These in vitro models closely resemble early embryos in terms of morphology and gene expression and provide a faithful recapitulation of early pre- and post-implantation embryonic development. Synthetic embryos can be generated through a combinatorial culture of three blastocyst-derived stem cell types, such as embryonic stem cells, trophoblast stem cells, and extraembryonic endoderm cells, or totipotent-like stem cells alone. This review provides an overview of the progress and various approaches in studying in vitro embryogenesis and gastrulation in mice and humans using stem cells. Furthermore, recent findings and breakthroughs in synthetic embryos and gastruloids are outlined. Despite ethical considerations, synthetic embryo models hold promise for understanding mammalian (including humans) embryonic development and have potential implications for regenerative medicine and developmental research. [ABSTRACT FROM AUTHOR]

Background: The development of the brain requires precise coordination of molecular processes across many cell-types. Underpinning these events are gene expression programs which require intricate regulation by non-coding regulatory sequences known as enhancers. In the context of the developing brain, transcribed enhancers (TEs) regulate temporally-specific expression of genes critical for cell identity and differentiation. Transcription of non-coding RNAs at active enhancer sequences, known as enhancer RNAs (eRNAs), is tightly associated with enhancer activity and has been correlated with target gene expression. TEs have been characterized in a multitude of developing tissues, however their regulatory role has yet to be described in the context of embryonic and early postnatal brain development. In this study, eRNA transcription was analyzed to identify TEs active during cerebellar development, as a proxy for the developing brain. Cap Analysis of Gene Expression followed by sequencing (CAGE-seq) was conducted at 12 stages throughout embryonic and early postnatal cerebellar development. Results: Temporal analysis of eRNA transcription identified clusters of TEs that peak in activity during either embryonic or postnatal times, highlighting their importance for temporally specific developmental events. Functional analysis of putative target genes identified molecular mechanisms under TE regulation revealing that TEs regulate genes involved in biological processes specific to neurons. We validate enhancer activity using in situ hybridization of eRNA expression from TEs predicted to regulate Nfib, a gene critical for cerebellar granule cell differentiation. Conclusions: The results of this analysis provide a valuable dataset for the identification of cerebellar enhancers and provide insight into the molecular mechanisms critical for brain development under TE regulation. This dataset is shared with the community through an online resource (https://goldowitzlab.shinyapps.io/trans-enh-app/). [ABSTRACT FROM AUTHOR]

Breast cancer is a major cause of cancer‐related morbidity and mortality in women. Estrogen receptor‐positive breast cancer accounts for roughly 70%‐80% of breast tumors, and estrogen receptor alpha (ERα) has been considered as a key driver in promoting breast cancer progression. In the present study, we identified USP37 as a novel modulator in modulating ERα ubiquitination and stability. The expression of USP37 was upregulated in ERα‐positive breast cancer and correlated with ERα protein level. High expression of USP37 was associated with unfavorable prognosis. USP37 depletion resulted in significantly decreased ERα protein level, ERα target genes expression as well as the estrogen response element activity in breast cancer cells. Further mechanistic study revealed the interaction between USP37 and ERα: USP37 regulated ERα signaling through modulating protein stability instead of gene expression, in which it stabilized ERα protein via inhibiting the K48‐specific polyubiquitination process. Additionally, USP37 depletion led to growth inhibition and cell cycle arrest of ERα‐positive breast cancer cells, which could be further rescued by ERα overexpression. Overall, our study proposed a novel post‐translational mechanism of ERα in promoting breast cancer progression. Targeting USP37 may be proved to be a promising strategy for patients with ERα‐positive breast cancer. [ABSTRACT FROM AUTHOR]

Background: Y-box binding protein 1 (YBX1) is a multifunctional oncoprotein that can interact with several long non-coding RNAs (lncRNAs) to regulate metastasis in malignancies including breast cancer (BC). In the present study, we demonstrated the association of YBX1 with oncogenic lncRNA SBF2-AS1 (SET-binding factor 2 antisense RNA 1) via PI3K/AKT/mTOR signaling to regulate BC cell proliferation. We further explored the involvement of the YBX1/SBF2-AS1/PI3K/AKT/mTOR axis in the restoration of tamoxifen (TAM) sensitivity. Methods and results: YBX1-SBF2-AS1 association was predicted in silico and verified by RNA immunoprecipitation (RIP)-qPCR assay. Transfection experiments, Real-time RT PCR, Western blots, Phospho AKT/mTOR antibody array kit, and cell proliferation/apoptosis assays were employed to detect the YBX1/SBF2-AS1/ PI3K/AKT/mTOR axis and its effects upon TAM treatment in vitro. We identified that the YBX1 protein specifically binds to lncRNA SBF2-AS1. Our transfection experiments in MCF-7 and MDA-MB-468 cells with SBF2-AS1 silenced or overexpressed YBX1 plasmids, and their negative controls revealed that YBX1 regulates the expression of SBF2-AS1 by forming a positive feedback loop for its activation. We further demonstrated YBX1-SBF2-AS1 association exerts its effects on cell proliferation via PI3K/AKT/mTOR signaling pathway. Furthermore, we observed an increase in TAM sensitivity in BC cells after the knockdown of YBX1-SBF2-AS1 marked by decreased cell proliferation through disruption of the PI3K/AKT/mTOR axis. Conclusion: Our study has identified a novel YBX1/SBF2-AS1/PI3K/AKT/mTOR regulatory axis which may serve as a potential target to improve the effectiveness and efficacy of TAM treatment in BC. [ABSTRACT FROM AUTHOR]

In the light of the current food security crisis, food adulteration has resurfaced on the international scene, inflicting potential safety issues and leading more and more consumers into deception. This situation led food control actors to remobilise their potential to face this problem, particularly in terms of analytical chemistry competencies. Similar to honey, grape molasses may be considered very likely to be adulterated leading to quality and authenticity issues, especially in the Eastern Mediterranean, where it is widely consumed as a traditional sweetener. This work reports the use of attenuated total reflectance–mid-infrared spectroscopy (ATR–MIR) coupled to chemometrics, as an alternative to complex, expensive and time-consuming analytical techniques, in the aim of detecting fraudulent glucose, fructose, sucrose and apple molasses additions to pure grape molasses. After collecting a widespread unadulterated grape molasses database, spiked samples with increasing concentrations (w/w) of the selected adulterants were prepared. In order to establish a qualitative model, whose potential is to detect adulteration and discriminate between the different adulterants, samples underwent ATR–MIR analyses without any prior preparation, and the collected spectral data were subjected to independent components analysis (ICA), where Random_ICA was used to retrieve the optimal number of independent components (ICs). Thereupon, the extraction of seven ICs allowed the establishment of a qualitative model with a clear discrimination between molasses adulterated with fructose, sucrose and glucose syrup, relying on MIR specific signals and incorporated ratios of the different adulterants. However, it failed in detecting apple molasses adulteration, calling for the development of a different analytical approach. The developed model underwent a verification step using a control set recorded on a different spectrometer, proving its potential to provide reproducible discrimination and classification rates. [ABSTRACT FROM AUTHOR]

Expansion microscopy (ExM) has been widely used to detect biomolecules in cultured cells and tissue samples due to its enablement of super resolution imaging with conventional microscopes, via physical expansion of samples. However, reaction conditions inherent to the process bring about strong fluorescent signal loss during polymerization and digestion and thus limit the brightness of the signal obtained post expansion. Here, we explore the impact of stabilizer‐containing organic fluorophores in ExM, as a mitigation strategy for this radical‐induced dye degradation. Through direct conjugation of 4‐nitrophenylalanine (NPA) to our previously developed trifunctional reagents, we validate and demonstrate that these multifunctional linkers enable visualization of different organelles with improved fluorescent intensity, owning to protection of the dyes to radical induced degradation as well as to photoprotection upon imaging. At this point, we cannot disentangle the relative contribution of both mechanisms. Furthermore, we report anchoring linkers that allow straightforward application of NPA or Trolox to commercially available fluorophore‐conjugated antibodies. We show that these anchoring linkers enable complete retention of biological targets while increasing fluorophore photostability. Our results provide guidance in exploring these stabilizer‐modified agents in ExM and methods for increased signal survival through the polymerization steps of the ExM protocols. [ABSTRACT FROM AUTHOR]

Menopausal hormone therapy (MHT) was widely used to treat menopause-related symptoms in menopausal women. However, MHT therapies were controversial with the increased risk of breast cancer because of different estrogen and progestogen combinations, and the molecular basis behind this phenomenon is currently not understood. To address this issue, we identified differentially expressed genes (DEGs) between the estrogen plus progestogens treatment (EPT) and estrogen treatment (ET) using the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) data. As a result, a total of 96 upregulated DEGs were first identified. Seven DEGs related to the cell cycle (CCNE2, CDCA5, RAD51, TCF19, KNTC1, MCM10, and NEIL3) were validated by RT-qPCR. Specifically, these seven DEGs were increased in EPT compared to ET (p < 0.05) and had higher expression levels in breast cancer than adjacent normal tissues (p < 0.05). Next, we found that estrogen receptor (ER)-positive breast cancer patients with a higher CNNE2 expression have a shorter overall survival time (p < 0.05), while this effect was not observed in the other six DEGs (p > 0.05). Interestingly, the molecular docking results showed that CCNE2 might bind to 17β-estradiol (−6.791 kcal/mol), progesterone (−6.847 kcal/mol), and medroxyprogesterone acetate (−6.314 kcal/mol) with a relatively strong binding affinity, respectively. Importantly, CNNE2 protein level could be upregulated with EPT and attenuated by estrogen receptor antagonist, acolbifene and had interactions with cancer driver genes (AKT1 and KRAS) and high mutation frequency gene (TP53 and PTEN) in breast cancer patients. In conclusion, the current study showed that CCNE2, CDCA5, RAD51, TCF19, KNTC1, MCM10, and NEIL3 might contribute to EPT-related tumorigenesis in breast cancer, with CCNE2 might be a sensitive risk indicator of breast cancer risk in women using MHT. [ABSTRACT FROM AUTHOR]

Enhancers in higher eukaryotes and upstream activating sequences (UASs) in yeast have been shown to recruit components of the RNA polymerase II (Pol II) transcription machinery. At least a fraction of Pol II recruited to enhancers in higher eukaryotes initiates transcription and generates enhancer RNA (eRNA). In contrast, UASs in yeast do not recruit transcription factor TFIIH, which is required for transcription initiation. For both yeast and mammalian systems, it was shown that Pol II is transferred from enhancers/UASs to promoters. We propose that there are two modes of Pol II recruitment to enhancers in higher eukaryotes. Pol II complexes that generate eRNAs are recruited via TFIID, similar to mechanisms operating at promoters. This may involve the binding of TFIID to acetylated nucleosomes flanking the enhancer. The resulting eRNA, together with enhancer-bound transcription factors and co-regulators, contributes to the second mode of Pol II recruitment through the formation of a transcription initiation domain. Transient contacts with target genes, governed by proteins and RNA, lead to the transfer of Pol II from enhancers to TFIID-bound promoters. [ABSTRACT FROM AUTHOR]

AMEERA-1 is a Phase 1/2 open-label single-arm study evaluating once-daily (QD) amcenestrant, an orally bioavailable selective estrogen receptor (ER) degrader, in postmenopausal women with ER+/HER2− advanced breast cancer (NCT03284957), who were mostly heavily pretreated (including targeted therapies and fulvestrant). In the dose escalation phase (Part A: n = 16), patients received amcenestrant 20-600 mg QD. Based on absence of dose-limiting toxicities, paired functional 18F-fluoroestradiol positron emission tomography, and pharmacokinetics, 400 mg QD was selected as recommended Phase 2 dose (RP2D) for the dose expansion phase (Part B: n = 49). No Grade ≥3 treatment-related adverse events or clinically significant cardiac/eye toxicities were reported. The Part B primary endpoint, confirmed objective response rate (ORR) was 3/45 at the interim analysis and 5/46 (10.9%) at the final analysis. The overall clinical benefit rate (CBR) was 13/46 (28.3%). CBRs among patients with baseline wild-type and mutated ESR1 were 9/26 (34.6%) and 4/19 (21.1%), respectively. Paired tumor biopsy and cell-free DNA analyses revealed ER inhibition and degradation, and a reduction in detectable ESR1 mutations, including Y537S. In conclusion, amcenestrant at RP2D of 400 mg QD for monotherapy is well-tolerated with no dose-limiting toxicities, and demonstrates preliminary antitumor activity irrespective of baseline ESR1 mutation status. There is a need for potent and non-toxic estrogen receptor (ER) antagonists to overcome the limitations of existing endocrine therapies. Here the authors report the results from Arm 1 of the Phase 1/2 study (AMEERA-1) among postmenopausal women with ER+/HER2− advanced breast cancer, which evaluates the safety, antitumor activity, pharmacokinetics, and pharmacodynamics of amcenestrant administered as monotherapy. [ABSTRACT FROM AUTHOR]

Background: In spite of the significant escalation in the depth of our conception and regulation of breast cancer over the past decades, the malady is still a serious community health challenge globally and poses a substantial tasks. Selective estrogen modulators (SERMs) such as Tamoxifen are approved for the therapy of this illness but developed drug resistance and unwanted side effects such as endometrial cancer caused by the long-term Tamoxifen chemotherapy limit their therapeutic applicability. Hence, developing new ER+ drugs with better therapeutic effect is strongly needed. In an attempt to overcome this challenge, this research is aimed at designing novel chromen-2-one analogues with better inhibition capacity against MCF-7 breast cancer cell line via structural modification of the reference compound and predict their activities using a developed QSAR model. Results: Four models were developed, and the first was selected for the design as it has the highest statistical parameters such as: coefficient of determination (R2 = 0.950), cross-validation coefficient (Qcv2 = 0.912), adjusted R2 (Radj2 = 0.935), and external validation R2 (Rpred2 = 0.7485). Twelve (12) new novel chromen-2-one analogs were designed through structural modification of the reference compound. Their activities was predicted using the selected model, and their pIC50 was found to be better than that of the reference compound and standard drug (Tamoxifen) used in the research. Results of pharmacokinetic study of the designed compounds revealed that they possess drug-likeness properties as none of them violated the Lipinski's rule of five while ADMET studies confirmed designed compounds 6, 8, 11 and 12 as orally safe and non-toxic. Furthermore, molecular docking analysis was performed between these orally safe designed compounds and the active site of the ER+ receptor and the result showed that they have higher binding affinities than the reference compound and the standard drug used for this research. Conclusion: Hence, designed compounds 6, 8, 11 and 12 can be used as novel ER+ breast cancer drug candidates after performing in vivo and in vitro studies. [ABSTRACT FROM AUTHOR]

ZB716 is a synthetic, steroidal, orally active anti-estrogen agent that is under clinical development for the treatment of estrogen receptor (ER)-positive metastatic breast cancer. The stable isotope-labeled ZB716 was required for use as an internal standard in LC-MS/MS assays. Therefore, a novel deuterated ZB716 (ZB716-d6) as an isotopically labeled internal standard was designed and synthesized through a newly developed route, which prepared ZB716-d6 in eight steps from the commercially available deuterium-labeled starting material [2H6]pentafluoropentanol. This procedure is very practicable and gives the final compound in good yield (19% total yield) and high purity (D, >99%, chemical purity 98%). At present, ZB716-d6 has been successfully used as an internal standard in clinical bioanalysis. [ABSTRACT FROM AUTHOR]

Despite all precautionary actions and the possibility of using vaccinations to counteract infections caused by human papillomaviruses (HPVs), HPV-related cancers still account for approximately 5% of all carcinomas. Worldwide, many women are still excluded from adequate health care due to their social position and origin. Therefore, immense efforts in research and therapy are still required to counteract the challenges that this disease entails. The special thing about an HPV infection is that it is not only able to trick the immune system in a sophisticated way, but also, through genetic integration into the host genome, to use all the resources available to the host cells to complete the replication cycle of the virus without activating the alarm mechanisms of immune recognition and elimination. The mechanisms utilized by the virus are the metabolic, immune, and hormonal signaling pathways that it manipulates. Since the virus is dependent on replication enzymes of the host cells, it also intervenes in the cell cycle of the differentiating keratinocytes and shifts their terminal differentiation to the uppermost layers of the squamocolumnar transformation zone (TZ) of the cervix. The individual signaling pathways are closely related and equally important not only for the successful replication of the virus but also for the onset of cervical cancer. We will therefore analyze the effects of HPV infection on metabolic signaling, as well as changes in hormonal and immune signaling in the tumor and its microenvironment to understand how each level of signaling interacts to promote tumorigenesis of cervical cancer. [ABSTRACT FROM AUTHOR]

Chromosomal rearrangements leading to the relocation of proto-oncogenes into transcription-active regions are found in various types of tumors. In particular, the transfer of proto-oncogenes to the locus of heavy chains of immunoglobulins (IGH) is frequently observed in B-lymphomas. The increased expression of the MYC proto-oncogene due to IGH/MYC translocation is detected in approximately 85% of Burkitt lymphoma cases. The regulatory mechanisms affecting the oncogenes upon translocation include non-coding enhancer RNAs (eRNAs). We conducted a search for the eRNAs that may affect MYC transcription in the case of IGH/MYC translocation in Burkitt lymphoma, looking for potentially oncogenic eRNAs located at the IGH locus and predominantly expressed in B cells. Overexpression and knockdown of our primary candidate eRNA AL928768.3 led to the corresponding changes in the expression of MYC proto-oncogene in Burkitt lymphoma cells. Furthermore, we demonstrated that AL928768.3 knockdown decreased lymphoma cell proliferation and resistance to chemotherapy. Significant effects were observed only in cell lines bearing IGH/MYC abnormality but not in B-cell lines without this translocation nor primary B-cells. Our results indicate that AL928768.3 plays an important role in the development of Burkitt's lymphoma and suggest it and similar, yet undiscovered eRNAs as potential tissue-specific targets for cancer treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: In cancer, regulatory regions of the genome are hijacked by the tumor cells for the activation of oncogenes that lead to cancer initiation and progression. One of the most salient regulatory elements of the genome are called enhancers, which are characterized by their ability to increase the expression of their target genes. In this study, we identified a novel enhancer that drives the expression of the oncogene CSF1 in triple-negative breast cancer patients, the most aggressive subtype of breast cancer. We demonstrate that this enhancer is specifically active in triple-negative breast cancer patients compared to other breast cancer subtypes and show that its target genes portend a worse clinical outcome in patients. We then use innovative CRISPR-based genome engineering techniques to systematically perturb various features of this enhancer to elucidate its mechanisms of action and determine the consequences on tumor cell growth. Furthermore, we test our model for CSF1 enhancer function in ovarian cancer cells and demonstrate that our findings can apply to other cancer types. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention. Enhancers are critical regulatory elements in the genome that help orchestrate spatiotemporal patterns of gene expression during development and normal physiology. In cancer, enhancers are often rewired by various genetic and epigenetic mechanisms for the activation of oncogenes that lead to initiation and progression. A key feature of active enhancers is the production of non-coding RNA molecules called enhancer RNAs, whose functions remain unknown but can be used to specify active enhancers de novo. Using a combination of eRNA transcription and chromatin modifications, we have identified a novel enhancer located 30 kb upstream of Colony Stimulating Factor 1 (CSF1). Notably, CSF1 is implicated in the progression of breast cancer, is overexpressed in triple-negative breast cancer (TNBC) cell lines, and its enhancer is primarily active in TNBC patient tumors. Genomic deletion of the enhancer (via CRISPR/Cas9) enabled us to validate this regulatory element as a bona fide enhancer of CSF1 and subsequent cell-based assays revealed profound effects on cancer cell proliferation, colony formation, and migration. Epigenetic silencing of the enhancer via CRISPR-interference assays (dCas9-KRAB) coupled to RNA-sequencing, enabled unbiased identification of additional target genes, such as RSAD2, that are predictive of clinical outcome. Additionally, we repurposed the RNA-guided RNA-targeting CRISPR-Cas13 machinery to specifically degrade the eRNAs transcripts produced at this enhancer to determine the consequences on CSF1 mRNA expression, suggesting a post-transcriptional role for these non-coding transcripts. Finally, we test our eRNA-dependent model of CSF1 enhancer function and demonstrate that our results are extensible to other forms of cancer. Collectively, this work describes a novel enhancer that is active in the TNBC subtype, which is associated with cellular growth, and requires eRNA transcripts for proper enhancer function. These results demonstrate the significant impact of enhancers in cancer biology and highlight their potential as tractable targets for therapeutic intervention. [ABSTRACT FROM AUTHOR]

Luminal breast cancer, an etiologically heterogeneous disease, is characterized by high steroid hormone receptor activity and aberrant gene expression profiles. Endocrine therapy and chemotherapy are promising therapeutic approaches to mitigate breast cancer proliferation and recurrence. However, the treatment of therapy-resistant breast cancer is a major challenge. Recent studies on breast cancer etiology have revealed the critical roles of epigenetic factors in luminal breast cancer tumorigenesis and drug resistance. Tumorigenic epigenetic factor-induced aberrant chromatin dynamics dysregulate the onset of gene expression and consequently promote tumorigenesis and metastasis. Epigenetic dysregulation, a type of somatic mutation, is a high-risk factor for breast cancer progression and therapy resistance. Therefore, epigenetic modulators alone or in combination with other therapies are potential therapeutic agents for breast cancer. Several clinical trials have analyzed the therapeutic efficacy of potential epi-drugs for breast cancer and reported beneficial clinical outcomes, including inhibition of tumor cell adhesion and invasiveness and mitigation of endocrine therapy resistance. This review focuses on recent findings on the mechanisms of epigenetic factors in the progression of luminal breast cancer. Additionally, recent findings on the potential of epigenetic factors as diagnostic biomarkers and therapeutic targets for breast cancer are discussed. [ABSTRACT FROM AUTHOR]

Steroid receptors (SRs) are members of the nuclear hormonal receptor family, many of which are transcription factors regulated by ligand binding. SRs regulate various human physiological functions essential for maintenance of vital biological pathways, including development, reproduction, and metabolic homeostasis. In addition, aberrant expression of SRs or dysregulation of their signaling has been observed in a wide variety of pathologies. SR activity is tightly and finely controlled by post-translational modifications (PTMs) targeting the receptors and/or their coregulators. Whereas major attention has been focused on phosphorylation, growing evidence shows that methylation is also an important regulator of SRs. Interestingly, the protein methyltransferases depositing methyl marks are involved in many functions, from development to adult life. They have also been associated with pathologies such as inflammation, as well as cardiovascular and neuronal disorders, and cancer. This article provides an overview of SR methylation/demethylation events, along with their functional effects and biological consequences. An in-depth understanding of the landscape of these methylation events could provide new information on SR regulation in physiology, as well as promising perspectives for the development of new therapeutic strategies, illustrated by the specific inhibitors of protein methyltransferases that are currently available. [ABSTRACT FROM AUTHOR]

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. Ras-GAP SH3-domain-binding protein 1 (G3BP1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3' untranslated region. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data indicate that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease. [ABSTRACT FROM AUTHOR]

Sex profoundly affects cancer incidence and susceptibility to therapy, with sex hormones highly contributing to this disparity. Various studies and omics data suggest a relationship between sex and the oncosuppressor p53 circuitry, including its regulators MDM2 and MDM4. Association of this network with genetic variation underlies sex-related altered cancer risk, age of onset, and cancer sensitivity to therapy. Moreover, sex-related factors, mainly estrogenic hormones, can affect the levels and/or function of the p53 network both in hormone-dependent and independent cancer. Despite this evidence, preclinical and clinical studies aimed to evaluate p53 targeted therapy rarely consider sex and related factors. This review summarizes the studies reporting the relationship between sex and the p53 circuitry, including its associated regulators, MDM2 and MDM4, with particular emphasis on estrogenic hormones. Moreover, we reviewed the evaluation of sex/hormone in preclinical studies and clinical trials employing p53-target therapies, and discuss how patients' sex and hormonal status could impact these therapeutic approaches. [ABSTRACT FROM AUTHOR]

As the essential sexual hormone, estrogen and its receptor has been proved to participate in the regulation of autoimmunity diseases and anti-tumor immunity. The adjustment of tumor immunity is related to the interaction between cancer cells, immune cells and tumor microenvironment, all of which is considered as the potential target in estrogen-induced immune system regulation. However, the specific mechanism of estrogen-induced immunity is poorly understood. Typically, estrogen causes the nuclear localization of estrogen/estrogen receptor complex and alternates the transcription pattern of target genes, leading to the reprogramming of tumor cells and differentiation of immune cells. However, the estrogen-induced non-canonical signal pathway activation is also crucial to the rapid function of estrogen, such as NF-κB, MAPK-ERK, and β-catenin pathway activation, which has not been totally illuminated. So, the investigation of estrogen modulatory mechanisms in these two manners is vital for the tumor immunity and can provide the potential for endocrine hormone targeted cancer immunotherapy. Here, this review summarized the estrogen-induced canonical and non-canonical signal transduction pathway and aimed to focus on the relationship among estrogen and cancer immunity as well as immune-related tumor microenvironment regulation. Results from these preclinical researches elucidated that the estrogen-target therapy has the application prospect of cancer immunotherapy, which requires the further translational research of these treatment strategies. [ABSTRACT FROM AUTHOR]

Globally, human papilloma virus (HPV) infection is a common sexually transmitted disease. However, most of the HPV infections eventually resolve aided by the body's efficient cell-mediated immune responses. In the vast majority of the small group of patients who develop overt disease too, it is the immune response that culminates in regression of lesions. It is therefore a rarity that persistent infection by high-risk genotypes of HPV compounded by other risk factors progresses through precancer (various grades of cervical intraepithelial neoplasia—CIN) to cervical cancer (CxCa). Hence, although CxCa is a rare culmination of HPV infection, the latter is nevertheless causally linked to >90% of cancer. The three 'Es' of cancer immunoediting viz. elimination, equilibrium, and escape come into vogue during the gradual evolution of CIN 1 to CxCa. Both cell-intrinsic and extrinsic mechanisms operate to eliminate virally infected cells: cell-extrinsic players are anti-tumor/antiviral effectors like Th1 subset of CD4+ T cells, CD8+ cytotoxic T cells, Natural Killer cells, etc. and pro-tumorigenic/immunosuppressive cells like regulatory T cells (Tregs), Myeloid-Derived Suppressor Cells (MDSCs), type 2 macrophages, etc. And accordingly, when immunosuppressive cells overpower the effectors e.g., in high-grade lesions like CIN 2 or 3, the scale is tilted towards immune escape and the disease progresses to cancer. Estradiol has long been considered as a co-factor in cervical carcinogenesis. In addition to the gonads, the Peyer's patches in the gut synthesize estradiol. Over and above local production of the hormone in the tissues, estradiol metabolism by the gut microbiome: estrobolome versus tryptophan non-metabolizing microbiome, regulates free estradiol levels in the intestine and extraintestinal mucosal sites. Elevated tissue levels of the hormone serve more than one purpose: besides a direct growth-promoting action on cervical epithelial cells, estradiol acting genomically via Estrogen Receptor- α also boosts the function of the stromal and infiltrating immunosuppressive cells viz. Tregs, MDSCs, and carcinoma-associated fibroblasts. Hence as a corollary, therapeutic repurposing of Selective Estrogen Receptor Disruptors or aromatase inhibitors could be useful for modulating immune function in cervical precancer/cancer. The immunomodulatory role of estradiol in HPV-mediated cervical lesions is reviewed. [ABSTRACT FROM AUTHOR]

Enhancers are well established as critical regulators of gene expression, but the mechanisms underlying the molecular basis of their specificity and activity are only partly understood. One of the most exciting recent observations is the discovery of enhancer RNA (eRNA), a class of noncoding RNAs derived from enhancer regions. Transcription of developmentally regulated enhancers has been observed to be associated with their active state. The nature of transcripts (eRNA) and their functional attributes are diverse and context dependent. The majority of eRNA are nonpolyadenylated and present in low abundance owing to their low stability, and may represent transcriptional noise. However, some eRNAs have been reported to be reasonably long and stable, are enriched in nuclei, exhibit tissue‐specific expression and may contribute to enhancer function. Transcription of enhancers has been postulated to mediate enhancer function through either the act of transcription or via the transcribed RNA per se and is a useful feature to be analysed to understand mechanisms underlying enhancer activity. Enhancer Eβ at the murine TCRβ locus has been reported to exhibit enhanced occupancy of RNA polymerase II in developing thymocytes. Here, we investigated the transcriptional potential of Eβ in developing thymocytes and detected overlapping bidirectional transcripts at Eβ ranging between 0.7 and 1.7 kb. These noncoding transcripts are capped, polyadenylated, nuclear and expressed specifically in thymocytes. Delineation of these characteristics is important to further investigate their functional roles in mediating enhancer activity. [ABSTRACT FROM AUTHOR]

Breast cancer is the most common type of cancer that affects females globally. Radiotherapy is a standard treatment option for breast cancer, where one of its most significant limitations is radioresistance development. MicroRNAs (miRNAs) are small, non-protein-coding RNAs that have been widely studied for their roles as disease biomarkers. To date, several in vitro, in vivo, and clinical studies have reported the roles of miRNAs in regulating radiosensitivity and radioresistance in breast cancer cells. This article reviews the roles of miRNAs in regulating treatment response toward radiotherapy and the associating cellular pathways. We identified 36 miRNAs that play a role in mediating radio-responses; 22 were radiosensitizing, 12 were radioresistance-promoting, and two miRNAs were reported to promote both effects. A brief overview of breast cancer therapy options, mechanism of action of radiation, and molecular mechanism of radioresistance was provided in this article. A summary of the latest clinical researches involving miRNAs in breast cancer radiotherapy was also included. [ABSTRACT FROM AUTHOR]

It has long been observed that human protein‐coding genes have a particular distribution of GC‐content: the 5′ end of these genes has high GC‐content while the 3′ end has low GC‐content. In 2012, it was proposed that this pattern of GC‐content could act as an mRNA identity feature that would lead to it being better recognized by the cellular machinery to promote its nuclear export. In contrast, junk RNA, which largely lacks this feature, would be retained in the nucleus and targeted for decay. Now two recent papers have provided evidence that GC‐content does promote the nuclear export of many mRNAs in human cells. [ABSTRACT FROM AUTHOR]

Endocrine therapies with SERMs (selective estrogen receptor modulators) or SERDs (selective estrogen receptor downregulators) are standard therapies for patients with estrogen receptor (ER)-positive breast cancer. Multiple small molecule inhibitors targeting the PI3K-AKT-mTOR pathway or CDK4/6 have been developed to be used in combination with anti-estrogen drugs to overcome endocrine resistance. In addition to their direct antitumor effects, accumulating evidence has revealed the tumor immune microenvironment (TIM)-modulating effects of these therapeutic strategies, which have not been properly acknowledged previously. The immune microenvironment of breast tumors plays a crucial role in tumor development, metastasis and treatment response to endocrine therapy and immunotherapy. Therefore, in our current work, we comprehensively review the immunomodulatory effect of endocrine therapy and discuss its potential applications in combination with immune checkpoint inhibitors in breast cancer treatment. [ABSTRACT FROM AUTHOR]

Gene expression and regulation play diverse and important roles across all living systems. By quantifying the expression, whether in a sample of single cells, a specific tissue, or in a whole animal, one can gain insights into the underlying biology. Many biological questions now require single-animal and tissue-specific resolution, such as why individuals, even within an isogenic population, have variations in development and aging across different tissues and organs. The popular techniques that quantify the transcriptome (e.g. RNA-sequencing) process populations of animals and cells together and thus, have limitations in both individual and spatial resolution. There are single-animal assays available (e.g. fluorescent reporters); however, they suffer other technical bottlenecks, such as a lack of robust sample-handling methods. Microfluidic technologies have demonstrated various improvements throughout the years, and it is likely they can enhance the impact of these single-animal gene-expression assays. In this perspective, we aim to highlight how the engineering/method-development field have unique opportunities to create new tools that can enable us to robustly answer the next set of important questions in biology that require high-density, high-quality gene expression data. [ABSTRACT FROM AUTHOR]

RNA, the transcriptional output of genomes, not only templates protein synthesis or directly engages in catalytic functions, but can feed back to the genome and serve as regulatory input for gene expression. Transcripts affecting the RNA abundance of other genes act by mechanisms similar to and in concert with protein factors that control transcription. Through recruitment or blocking of activating and silencing complexes to specific genomic loci, RNA and protein factors can favor transcription or lower the local gene expression potential. Most regulatory proteins enter nuclei from all directions to start the search for increased affinity to specific DNA sequences or to other proteins nearby genuine gene targets. In contrast, RNAs emerge from spatial point sources within nuclei, their encoding genes. A transcriptional burst can result in the local appearance of multiple nascent RNA copies at once, in turn increasing local nucleic acid density and RNA motif abundance before diffusion into the nuclear neighborhood. The confined initial localization of regulatory RNAs causing accumulation of protein co-factors raises the intriguing possibility that target specificity of non-coding, and probably coding, RNAs is achieved through gene/RNA positioning and spatial proximity to regulated genomic regions. Here we review examples of positional cis conservation of regulatory RNAs with respect to target genes, spatial proximity of enhancer RNAs to promoters through DNA looping and RNA-mediated formation of membrane-less structures to control chromatin structure and expression. We speculate that linear and spatial proximity between regulatory RNA-encoding genes and gene targets could possibly ease the evolutionary pressure on maintaining regulatory RNA sequence conservation. [ABSTRACT FROM AUTHOR]

Transcription in several organisms from certain bacteria to humans has been observed to be stochastic in nature: toggling between active and inactive states. Periods of active nascent RNA synthesis known as bursts represent individual gene activation events in which multiple polymerases are initiated. Therefore, bursting is the single locus illustration of both gene activation and repression. Although transcriptional bursting was originally observed decades ago, only recently have technological advances enabled the field to begin elucidating gene regulation at the single-locus level. In this review, we focus on how biochemical, genomic, and single-cell data describe the regulatory steps of transcriptional bursts. [ABSTRACT FROM AUTHOR]

Endocrine therapy resistance frequently develops in estrogen receptor positive (ER+) breast cancer, but the underlying molecular mechanisms are largely unknown. Here, we show that 3-dimensional (3D) chromatin interactions both within and between topologically associating domains (TADs) frequently change in ER+ endocrine-resistant breast cancer cells and that the differential interactions are enriched for resistance-associated genetic variants at CTCF-bound anchors. Ectopic chromatin interactions are preferentially enriched at active enhancers and promoters and ER binding sites, and are associated with altered expression of ER-regulated genes, consistent with dynamic remodelling of ER pathways accompanying the development of endocrine resistance. We observe that loss of 3D chromatin interactions often occurs coincidently with hypermethylation and loss of ER binding. Alterations in active A and inactive B chromosomal compartments are also associated with decreased ER binding and atypical interactions and gene expression. Together, our results suggest that 3D epigenome remodelling is a key mechanism underlying endocrine resistance in ER+ breast cancer. Endocrine therapy resistance occurs often in estrogen receptor positive (ER+) breast cancer. Here, the authors find that 3D epigenome remodelling at ER-bound enhancer-promoter interactions is a key mechanism underlying endocrine resistance. [ABSTRACT FROM AUTHOR]

Estrogen receptor positive breast neoplasias represent over 70% of diagnosed breast cancers. Depending on the stage at which the tumor is detected, HER2 status and genomic risk, endocrine therapy is combined with either radio, chemo and/or targeted therapy. A growing amount of evidence supports the notion that components of the tumor microenvironment play specific roles in response to treatment and that strategies targeting these key interactions with tumor cells could pave the way to a new generation of therapies. In this review, we analyze the evidence suggesting different components of the tumor microenvironment play a role in hormone receptor positive breast cancer progression. In particular we focus on the immune system, carcinoma associated fibroblasts and the extracellular matrix. Further insight into the cross talk between these constituents of the microenvironment and the tumor cells may lead to therapies that eliminate disseminated metastatic cells early on, and thus reduce distant disease relapse which is the leading cause of death for patients who are diagnosed with this illness. [ABSTRACT FROM AUTHOR]

In recent years, studies have shown that enhancer RNAs (eRNAs) can be transcribed from enhancers. Increasing evidence has revealed that eRNAs play critical roles in the development of various cancers. Oestrogen‐associated eRNAs are closely related to breast cancer. In view of the gender differences in bladder cancer (BCa), we suppose that oestrogen‐associated eRNAs are also involved in tumorigenesis of BCa. In our study, we first demonstrated that eGREB1 derived from the enhancer of an oestrogen‐responsive gene—GREB1 was up‐regulated in BCa tissues, and the expression level of eGREB1 is positively associated with the histological grade and TNM stage of BCa. Knockdown of eGREB1 by CRISPR‐Cas13a could inhibit cell proliferation, migration and invasion and induce apoptosis in BCa cells T24 and 5637. Besides, we exhibited the promoting effect of oestrogen on BCa cells. What's more, down‐regulation of eGREB1 could improve the malignant biological characteristics of BCa cells induced by oestrogen. In conclusion, our data indicated that eGREB1 plays oncogenic role and oestrogen may promote the occurrence and progression of BCa by inducing eGREB1 production. Our findings provide new insights into the prevention of BCa and develop a novel therapeutic target for the treatment of BCa. [ABSTRACT FROM AUTHOR]