Clonal Origin and Lineage Ambiguity in Mixed Neuroendocrine Carcinoma of the Uterine Cervix.

Small-cell neuroendocrine carcinoma (SCNEC) of the cervix is a rare disease characterized by a high incidence of mixed tumors with other types of cancer. The mechanism underlying this mixed phenotype is not well understood. This study established a panel of organoid lines from patients with SCNEC of the cervix and ultimately focused on one line, which retained a mixed tumor phenotype, both in vitro and in vivo. Histologically, both organoids and xenograft tumors showed distinct differentiation into either SCNEC or adenocarcinoma in some regions and ambiguous differentiation in others. Tracking single cells indicated the existence of cells with bipotential differentiation toward SCNEC and adenocarcinomas. Single-cell transcriptional analysis identified three distinct clusters: SCNEC-like, adenocarcinoma-like, and a cluster lacking specific differentiation markers. The expression of neuroendocrine markers was enriched in the SCNEC-like cluster but not exclusively. Human papillomavirus 18 E6 was enriched in the SCNEC-like cluster, which showed higher proliferation and lower levels of the p53 pathway. After treatment with anticancer drugs, the expression of adenocarcinoma markers increased, whereas that of SCNEC decreased. Using a reporter system for keratin 19 expression, changes in the differentiation of each cell were shown to be associated with the shift in differentiation induced by drug treatment. These data suggest that mixed SCNEC/cervical tumors have a clonal origin and are characterized by an ambiguous and flexible differentiation state.

LncRNA ZNNT1 induces p53 degradation by interfering with the interaction between p53 and the SART3-USP15 complex.

Mammalian genomes encode large number of long noncoding RNAs (lncRNAs) that play key roles in various biological processes, including proliferation, differentiation, and stem cell pluripotency. Recent studies have addressed that some lncRNAs are dysregulated in human cancers and may play crucial roles in tumor development and progression. Here, we show that the lncRNA ZNNT1 is required for the proliferation and tumorigenicity of colon cancer cells with wild-type p53. ZNNT1 knockdown leads to decreased ubiquitination and stabilization of p53 protein. Moreover, we demonstrate that ZNNT1 needs to interact with SART3 to destabilize p53 and to promote the proliferation and tumorigenicity of colon cancer cells. We further show that SART3 is associated with the ubiquitin-specific peptidase USP15 and that ZNNT1 may induce p53 destabilization by inhibiting this interaction. These results suggest that ZNNT1 interferes with the SART3-USP15 complex-mediated stabilization of p53 protein and thereby plays important roles in the proliferation and tumorigenicity of colon cancer cells. Our findings suggest that ZNNT1 may be a promising molecular target for the therapy of colon cancer.

Essential Role of STAT3 Signaling in Hair Follicle Homeostasis.

Dominant-negative mutations associated with signal transducer and activator of transcription 3 (STAT3) signaling, which controls epithelial proliferation in various tissues, lead to atopic dermatitis in hyper IgE syndrome. This dermatitis is thought to be attributed to defects in STAT3 signaling in type 17 helper T cell　specification. However, the role of STAT3 signaling in skin epithelial cells remains unclear. We found that STAT3 signaling in keratinocytes is required to maintain skin homeostasis by negatively controlling the expression of hair follicle-specific keratin genes. These expression patterns correlated with the onset of dermatitis, which was observed in specific pathogen-free conditions but not in germ-free conditions, suggesting the involvement of Toll-like receptor-mediated inflammatory responses. Thus, our study suggests that STAT3-dependent gene expression in keratinocytes plays a critical role in maintaining the homeostasis of skin, which is constantly exposed to microorganisms.

The Number of Transcription Factors at an Enhancer Determines Switch-like Gene Expression.

NF-κB is a transcription factor that activates super enhancers (SEs) and typical enhancers (TEs) and triggers threshold and graded gene expression, respectively. However, the mechanisms by which NF-κB selectively participates in these enhancers remain unclear. Here we show using mouse primary B lymphocytes that SE activity simultaneously associates with chromatin opening and enriched NF-κB binding, resulting in a higher fold change and threshold expression upon B cell receptor (BCR) activation. The higher fold change results from longer DNA, whereas the threshold response is explained by synergy in DNA-NF-κB binding and is supported by the coexistence of PU.1 and NF-κB in a SE before cell stimulation. This model indicates that the pre-existing NF-κB functions as a seed and triggers its processive binding upon BCR activation. Our mathematical modeling of the single-cell transcriptome reveals an additional role for SEs in divergent clonal responses in B cells.

Translation-dependent unwinding of stem-loops by UPF1 licenses Regnase-1 to degrade inflammatory mRNAs.

Regnase-1-mediated mRNA decay (RMD), in which inflammatory mRNAs harboring specific stem-loop structures are degraded, is a critical part of proper immune homeostasis. Prior to initial translation, Regnase-1 associates with target stem-loops but does not carry out endoribonucleolytic cleavage. Single molecule imaging revealed that UPF1 is required to first unwind the stem-loops, thus licensing Regnase-1 to proceed with RNA degradation. Following translation, Regnase-1 physically associates with UPF1 using two distinct points of interaction: The Regnase-1 RNase domain binds to SMG1-phosphorylated residue T28 in UPF1; in addition, an intrinsically disordered segment in Regnase-1 binds to the UPF1 RecA domain, enhancing the helicase activity of UPF1. The SMG1-UPF1-Regnase-1 axis targets pioneer rounds of translation and is critical for rapid resolution of inflammation through restriction of the number of proteins translated by a given mRNA. Furthermore, small-molecule inhibition of SMG1 prevents RNA unwinding in dendritic cells, allowing post-transcriptional control of innate immune responses.

Hepatic posttranscriptional network comprised of CCR4-NOT deadenylase and FGF21 maintains systemic metabolic homeostasis.

Whole-body metabolic homeostasis is tightly controlled by hormone-like factors with systemic or paracrine effects that are derived from nonendocrine organs, including adipose tissue (adipokines) and liver (hepatokines). Fibroblast growth factor 21 (FGF21) is a hormone-like protein, which is emerging as a major regulator of whole-body metabolism and has therapeutic potential for treating metabolic syndrome. However, the mechanisms that control FGF21 levels are not fully understood. Herein, we demonstrate that FGF21 production in the liver is regulated via a posttranscriptional network consisting of the CCR4-NOT deadenylase complex and RNA-binding protein tristetraprolin (TTP). In response to nutrient uptake, CCR4-NOT cooperates with TTP to degrade AU-rich mRNAs that encode pivotal metabolic regulators, including FGF21. Disruption of CCR4-NOT activity in the liver, by deletion of the catalytic subunit CNOT6L, increases serum FGF21 levels, which ameliorates diet-induced metabolic disorders and enhances energy expenditure without disrupting bone homeostasis. Taken together, our study describes a hepatic CCR4-NOT/FGF21 axis as a hitherto unrecognized systemic regulator of metabolism and suggests that hepatic CCR4-NOT may serve as a target for devising therapeutic strategies in metabolic syndrome and related morbidities.

Tamoxifen mechanically reprograms the tumor microenvironment via HIF-1A and reduces cancer cell survival.

The tumor microenvironment is fundamental to cancer progression, and the influence of its mechanical properties is increasingly being appreciated. Tamoxifen has been used for many years to treat estrogen-positive breast cancer. Here we report that tamoxifen regulates the level and activity of collagen cross-linking and degradative enzymes, and hence the organization of the extracellular matrix, via a mechanism involving both the G protein-coupled estrogen receptor (GPER) and hypoxia-inducible factor-1 alpha (HIF-1A). We show that tamoxifen reduces HIF-1A levels by suppressing myosin-dependent contractility and matrix stiffness mechanosensing. Tamoxifen also downregulates hypoxia-regulated genes and increases vascularization in PDAC tissues. Our findings implicate the GPER/HIF-1A axis as a master regulator of peri-tumoral stromal remodeling and the fibrovascular tumor microenvironment and offer a paradigm shift for tamoxifen from a well-established drug in breast cancer hormonal therapy to an alternative candidate for stromal targeting strategies in PDAC and possibly other cancers.

Hunt for the tipping point during endocrine resistance process in breast cancer by dynamic network biomarkers.

Acquired drug resistance is the major reason why patients fail to respond to cancer therapies. It is a challenging task to determine the tipping point of endocrine resistance and detect the associated molecules. Derived from new systems biology theory, the dynamic network biomarker (DNB) method is designed to quantitatively identify the tipping point of a drastic system transition and can theoretically identify DNB genes that play key roles in acquiring drug resistance. We analyzed time-course mRNA sequence data generated from the tamoxifen-treated estrogen receptor (ER)-positive MCF-7 cell line, and identified the tipping point of endocrine resistance with its leading molecules. The results show that there is interplay between gene mutations and DNB genes, in which the accumulated mutations eventually affect the DNB genes that subsequently cause the change of transcriptional landscape, enabling full-blown drug resistance. Survival analyses based on clinical datasets validated that the DNB genes were associated with the poor survival of breast cancer patients. The results provided the detection for the pre-resistance state or early signs of endocrine resistance. Our predictive method may greatly benefit the scheduling of treatments for complex diseases in which patients are exposed to considerably different drugs and may become drug resistant.

High-Quality Overlapping Paired-End Reads for the Detection of A-to-I Editing on Small RNA.

Paired-end RNA sequencing (RNA-seq) is usually applied to the quantification of long transcripts such as messenger or long non-coding RNAs, in which case overlapping pairs are discarded. In contrast, RNA-seq on short RNAs (≤ 200 nt) is typically carried out in single-end mode, as the additional cost associated with paired-end would only translate into redundant sequence information. Here, we exploit paired-end sequencing of short RNAs as a strategy to filter out sequencing errors and apply this method to the identification of adenosine-to-inosine (A-to-I) RNA editing events on human precursor microRNA (pre-miRNA) and mature miRNA. Combined with RNA immunoprecipitation sequencing (RIP-seq) of A-to-I RNA editing enzymes, this method takes full advantage of deep sequencing technology to identify RNA editing sites with unprecedented resolution in terms of editing efficiency.

Base-pairing probability in the microRNA stem region affects the binding and editing specificity of human A-to-I editing enzymes ADAR1-p110 and ADAR2.

Adenosine deaminases acting on RNA (ADARs) catalyze the deamination of adenosine (A) to inosine (I). A-to-I RNA editing targets double-stranded RNA (dsRNA), and increases the complexity of gene regulation by modulating base pairing-dependent processes such as splicing, translation, and microRNA (miRNA)-mediated gene silencing. This study investigates the genome-wide binding preferences of the nuclear constitutive isoforms ADAR1-p110 and ADAR2 on human miRNA species by RNA immunoprecipitation of ADAR-bound small RNAs (RIP-seq). Our results suggest that secondary structure predicted by base-pairing probability in the mainly double-stranded region of a pre-miRNA or mature miRNA duplex may determine ADAR isoform preference for binding distinct subpopulations of miRNAs. Furthermore, we identify 31 unique editing sites with statistical significance, 19 sites of which are novel editing sites. Editing sites are enriched in the seed region responsible for target recognition by miRNAs, and isoform-specific nucleotide motifs in the immediate vicinity and opposite of editing sites are consistent with previous studies, and further reveal that ADAR2 may edit A/C bulges more frequently than ADAR1-p110 in the context of miRNA.

Transcriptionally inducible Pleckstrin homology-like domain, family A, member 1, attenuates ErbB receptor activity by inhibiting receptor oligomerization.

Feedback control is a key mechanism in signal transduction, intimately involved in regulating the outcome of the cellular response. Here, we report a novel mechanism by which PHLDA1, Pleckstrin homology-like domain, family A, member 1, negatively regulates ErbB receptor signaling by inhibition of receptor oligomerization. We have found that the ErbB3 ligand, heregulin, induces PHILDA1 expression in MCF-7 cells. Transcriptionally-induced PHLDA1 protein directly binds to ErbB3, whereas knockdown of PHLDA1 increases complex formation between ErbB3 and ErbB2. To provide insight into the mechanism for our time-course and single-cell experimental observations, we performed a systematic computational search of network topologies of the mathematical models based on receptor dimer-tetramer formation in the ErbB activation processes. Our results indicate that only a model in which PHLDA1 inhibits formation of both dimers and tetramer can explain the experimental data. Predictions made from this model were further validated by single-molecule imaging experiments. Our studies suggest a unique regulatory feature of PHLDA1 to inhibit the ErbB receptor oligomerization process and thereby control the activity of receptor signaling network.

Genome-scale regression analysis reveals a linear relationship for promoters and enhancers after combinatorial drug treatment.

MOTIVATION: Drug combination therapy for treatment of cancers and other multifactorial diseases has the potential of increasing the therapeutic effect, while reducing the likelihood of drug resistance. In order to reduce time and cost spent in comprehensive screens, methods are needed which can model additive effects of possible drug combinations. RESULTS: We here show that the transcriptional response to combinatorial drug treatment at promoters, as measured by single molecule CAGE technology, is accurately described by a linear combination of the responses of the individual drugs at a genome wide scale. We also find that the same linear relationship holds for transcription at enhancer elements. We conclude that the described approach is promising for eliciting the transcriptional response to multidrug treatment at promoters and enhancers in an unbiased genome wide way, which may minimize the need for exhaustive combinatorial screens. AVAILABILITY AND IMPLEMENTATION: The CAGE sequence data used in this study is available in the DDBJ Sequence Read Archive (http://trace.ddbj.nig.ac.jp/index_e.html), accession number DRP001113. CONTACT: xin.gao@kaust.edu.sa or erik.arner@riken.jp. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An integrated expression atlas of miRNAs and their promoters in human and mouse.

MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.

Robustness analysis of the detailed kinetic model of an ErbB signaling network by using dynamic sensitivity.

The ErbB receptor signaling pathway plays an important role in the regulation of cellular proliferation, survival and differentiation, and dysregulation of the pathway is linked to various types of human cancer. Mathematical models have been developed as a practical complementary approach to deciphering the complexity of ErbB receptor signaling and elucidating how the pathways discriminate between ligands to induce different cell fates. In this study, we developed a simulator to accurately calculate the dynamic sensitivity of extracellular-signal-regulated kinase (ERK) activity (ERK*) and Akt activity (Akt*), downstream of the ErbB receptors stimulated with epidermal growth factor (EGF) and heregulin (HRG). To demonstrate the feasibility of this simulator, we estimated how the reactions critically responsible for ERK* and Akt* change with time and in response to different doses of EGF and HRG, and predicted that only a small number of reactions determine ERK* and Akt*. ERK* increased steeply with increasing HRG dose until saturation, while showing a gently rising response to EGF. Akt* had a gradual wide-range response to HRG and a blunt response to EGF. Akt* was sensitive to perturbations of intracellular kinetics, while ERK* was more robust due to multiple, negative feedback loops. Overall, the simulator predicted reactions that were critically responsible for ERK* and Akt* in response to the dose of EGF and HRG, illustrated the response characteristics of ERK* and Akt*, and estimated mechanisms for generating robustness in the ErbB signaling network.

Mathematical modeling of atopic dermatitis reveals "double-switch" mechanisms underlying 4 common disease phenotypes.

BACKGROUND: The skin barrier acts as the first line of defense against constant exposure to biological, microbial, physical, and chemical environmental stressors. Dynamic interplay between defects in the skin barrier, dysfunctional immune responses, and environmental stressors are major factors in the development of atopic dermatitis (AD). A systems biology modeling approach can yield significant insights into these complex and dynamic processes through integration of prior biological data. OBJECTIVE: We sought to develop a multiscale mathematical model of AD pathogenesis that describes the dynamic interplay between the skin barrier, environmental stress, and immune dysregulation and use it to achieve a coherent mechanistic understanding of the onset, progression, and prevention of AD. METHODS: We mathematically investigated synergistic effects of known genetic and environmental risk factors on the dynamic onset and progression of the AD phenotype, from a mostly asymptomatic mild phenotype to a severe treatment-resistant form. RESULTS: Our model analysis identified a "double switch," with 2 concatenated bistable switches, as a key network motif that dictates AD pathogenesis: the first switch is responsible for the reversible onset of inflammation, and the second switch is triggered by long-lasting or frequent activation of the first switch, causing irreversible onset of systemic TH2 sensitization and worsening of AD symptoms. CONCLUSIONS: Our mathematical analysis of the bistable switch predicts that genetic risk factors decrease the threshold of environmental stressors to trigger systemic TH2 sensitization. This analysis predicts and explains 4 common clinical AD phenotypes from a mild and reversible phenotype through to severe and recalcitrant disease and provides a mechanistic explanation for clinically demonstrated preventive effects of emollient treatments against development of AD.

Protective neutralizing influenza antibody response in the absence of T follicular helper cells.

Virus infection induces the development of T follicular helper (TFH) and T helper 1 (TH1) cells. Although TFH cells are important in anti-viral humoral immunity, the contribution of TH1 cells to a protective antibody response remains unknown. We found that IgG2 antibodies predominated in the response to vaccination with inactivated influenza A virus (IAV) and were responsible for protective immunity to lethal challenge with pathogenic H5N1 and pandemic H1N1 IAV strains, even in mice that lacked TFH cells and germinal centers. The cytokines interleukin-21 and interferon-γ, which are secreted from TH1 cells, were essential for the observed greater persistence and higher titers of IgG2 protective antibodies. Our results suggest that TH1 induction could be a promising strategy for producing effective neutralizing antibodies against emerging influenza viruses.

ASBEL-TCF3 complex is required for the tumorigenicity of colorectal cancer cells.

Wnt/ß-catenin signaling plays a key role in the tumorigenicity of colon cancer. Furthermore, it has been reported that lncRNAs are dysregulated in several steps of cancer development. Here we show that ß-catenin directly activates the transcription of the long noncoding RNA (lncRNA) ASBEL [antisense ncRNA in the ANA (Abundant in neuroepithelium area)/BTG3 (B-cell translocation gene 3) locus] and transcription factor 3 (TCF3), both of which are required for the survival and tumorigenicity of colorectal cancer cells. ASBEL interacts with and recruits TCF3 to the activating transcription factor 3 (ATF3) locus, where it represses the expression of ATF3. Furthermore, we demonstrate that ASBEL-TCF3-mediated down-regulation of ATF3 expression is required for the proliferation and tumorigenicity of colon tumor cells. ATF3, in turn, represses the expression of ASBEL Our results reveal a pathway involving an lncRNA and two transcription factors that plays a key role in Wnt/ß-catenin-mediated tumorigenesis. These results may provide insights into the variety of biological and pathological processes regulated by Wnt/ß-catenin signaling.

MYU, a Target lncRNA for Wnt/c-Myc Signaling, Mediates Induction of CDK6 to Promote Cell Cycle Progression.

Aberrant activation of Wnt/ß-catenin signaling is a major driving force in colon cancer. Wnt/ß-catenin signaling induces the expression of the transcription factor c-Myc, leading to cell proliferation and tumorigenesis. c-Myc regulates multiple biological processes through its ability to directly modulate gene expression. Here, we identify a direct target of c-Myc, termed MYU, and show that MYU is upregulated in most colon cancers and required for the tumorigenicity of colon cancer cells. Furthermore, we demonstrate that MYU associates with the RNA binding protein hnRNP-K to stabilize CDK6 expression and thereby promotes the G1-S transition of the cell cycle. These results suggest that the MYU/hnRNP-K/CDK6 pathway functions downstream of Wnt/c-Myc signaling and plays a critical role in the proliferation and tumorigenicity of colon cancer cells.

Stimulus-Dependent Inhibitor of Apoptosis Protein Expression Prolongs the Duration of B Cell Signalling.

Different dynamic behaviours of signalling activity can induce distinct biological responses in a variety of cells. However, the molecular mechanisms that determine the dynamics of kinase activities in immune cells are not well understood. In this study, we showed that the duration of both IκB kinase (IKK) and extracellular signal-regulated kinase (ERK) activities in B cell receptor (BCR)- and CD40-signalling pathways in B cells were regulated by transcriptional feedback loops. We conducted a time-course transcriptome analysis after BCR or CD40 stimulation and identified the following four candidate genes as feedback regulators for IKK and ERK: inhibitor of apoptosis protein (IAP), TNF alpha-induced protein 3, dual-specificity phosphatase 5, and sprouty homolog 2. Quantitative experiments and mathematical modelling suggested that IAP inhibition shortened the duration of IKK and ERK activity following both BCR and CD40 pathway stimulation, indicating a positive role for IAP in B cell signalling. Furthermore, transient kinase activities induced by IAP blockage reduced the levels of delayed expression genes. Together, our findings suggest that IKK and ERK activity durations can be fine-tuned by the coordinated regulation of positive and negative transcriptional feedback and that these network properties determine the biological output of B cells.

Genetic Network Inference Using Hierarchical Structure.

Many methods for inferring genetic networks have been proposed, but the regulations they infer often include false-positives. Several researchers have attempted to reduce these erroneous regulations by proposing the use of a priori knowledge about the properties of genetic networks such as their sparseness, scale-free structure, and so on. This study focuses on another piece of a priori knowledge, namely, that biochemical networks exhibit hierarchical structures. Based on this idea, we propose an inference approach that uses the hierarchical structure in a target genetic network. To obtain a reasonable hierarchical structure, the first step of the proposed approach is to infer multiple genetic networks from the observed gene expression data. We take this step using an existing method that combines a genetic network inference method with a bootstrap method. The next step is to extract a hierarchical structure from the inferred networks that is consistent with most of the networks. Third, we use the hierarchical structure obtained to assign confidence values to all candidate regulations. Numerical experiments are also performed to demonstrate the effectiveness of using the hierarchical structure in the genetic network inference. The improvement accomplished by the use of the hierarchical structure is small. However, the hierarchical structure could be used to improve the performances of many existing inference methods.