Cytoplasmic Signaling Cascades Research Articles

Oxidative stress in the skin: Impact and related protection.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Shaping Microglial Phenotypes Through Estrogen Receptors: Relevance to Sex-Specific Neuroinflammatory Responses to Brain Injury and Disease.

In mammals, 17β-estradiol (E2), the primary endogenous estrogen, maintains normal central nervous system (CNS) function throughout life and influences brain responses to injury and disease. Estradiol-induced cellular changes are mediated through the activation of nuclear and extranuclear estrogen receptors (ERs), which include ERα, ERβ, and the G-protein coupled receptor, GPER1. ERs are widely expressed throughout the brain, acting as transcriptional effectors or rapidly initiating membrane and cytoplasmic signaling cascades in practically all brain cells including microglia, the resident immune cells of the CNS. Activation of ERs by E2 exerts potent anti-inflammatory effects through mechanisms involving the modification of microglial cell responses to acute or chronic brain injury. Recent studies suggest that microglial maturation is influenced by the internal gonadal hormone milieu and that their functions in the normal and diseased brain are sex specific. However, the role that each ER subtype plays in microglial development or in determining microglial function as the primary cellular defense mechanism against pathogens and injury remains unclear. This is partly due to the fact that most studies investigating the mechanisms by which E2-ER signaling modifies microglial cellular phenotypes have been restricted to one sex or age. This review examines the different in vivo and in vitro models used to study the direct and indirect regulation of microglial cell function by E2 through ERs. Ischemic stroke will be used as an example of a neurologic disease in which activation of ERs shapes microglial phenotypes in response to injury in a sex- and age-specific fashion. SIGNIFICANCE STATEMENT: As the primary immune sensors of central nervous system damage, microglia are important potential therapeutic targets. Estrogen receptor signaling modulates microglial responses to brain injury and disease in a sex- and age-specific fashion. Hence, investigating the molecular mechanisms by which estrogen receptors regulate and shape microglial functions throughout life may result in novel and effective therapeutic opportunities that are tailored for each sex and age.

The structural basis for membrane assembly of immunoreceptor signalling complexes

Immunoreceptors are TM complexes that consist of separate ligand-binding and signal-transducing modules. Mounting evidence suggests that interactions with the local environment may influence the architecture of these TM domains, which assemble via crucial sets of conserved ionisable residues, and also control the peripheral association of immunoreceptor tyrosine-based activation motifs (ITAMs) whose phosphorylation triggers cytoplasmic signalling cascades. We now report a molecular dynamics (MD) simulation study of the archetypal T cell receptor (TCR) and its cluster of differentiation 3 (CD3) signalling partners, along with the analogous DNAX-activation protein of 12kDa (DAP12)/natural killer group 2C (NKG2C) complex. Based on > 15μs of explicitly solvated, atomic-resolution sampling, we explore molecular aspects of immunoreceptor complex stability in different functionally relevant states. A novel alchemical approach is used to simulate the cytoplasmic CD3ε tail at different depths within lipid bilayer models, revealing that the conformation and cytoplasmic exposure of ITAMs are highly sensitive to local enrichment by different lipid species and to phosphorylation. Furthermore, simulations of the TCR and DAP12 TM domains in various states of oligomerisation suggest that, during the early stages of assembly, stable membrane insertion is facilitated by the interfacial lipid/solvent environment and/or partial ionisation of charged residues. Collectively, our results indicate that the architecture and mechanisms of signal transduction in immunoreceptor complexes are tightly regulated by interactions with the microenvironment.

Overexpression of SMYD3 in Ovarian Cancer is Associated with Ovarian Cancer Proliferation and Apoptosis via Methylating H3K4 and H4K20.

Background: Epigenetic regulation has been verified as a key mechanism in tumorigenesis. SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase, is a promising epigenetic therapeutic target and is overexpressed in numerous human tumors. SMYD3 can promote oncogenic progression by methylating lysines to integrate cytoplasmic kinase signaling cascades or by methylating histone lysines to regulate specific gene transcription. However, the exact role of SMYD3 in the progression of ovarian cancer is still unknown.Methods: Immunohistochemistry was employed to test SMYD3 expression in ovarian cancer tissues from clinical patients. CCK-8 assay, Real-time cell analysis (RTCA), colony formation assay, cell cycle and apoptosis tested by Flow cytometer were employed to test the effects of SMYD3 on cell proliferation and apoptosis in ovarian cancer cell lines. A PCR array was used to identify the downstream targets of SMYD3. And, PCR and Western blot were used to verify their expression. The binding of SMYD3 on the promoter of target genes were tested by ChIP assays. We also use nude mice subcutaneous tumor model and patient-derived xenograft (PDX) model to investigate the tumor promotive function of SMYD3 in vivo.Results: SMYD3 expression was higher in ovarian cancer tissues and cell lines than in normal ovarian epithelial tissue and human ovarian surface epithelial cells (HOSEpiC). After silencing SMYD3, the proliferation of ovarian cancer cells was significantly inhibited in vitro. In addition, the SMYD3-specific small-molecule inhibitor BCI-121 suppressed ovarian cancer cell proliferation. Downregulation of SMYD3 led to S phase arrest and increased the cell apoptosis rate. Furthermore, a PCR array revealed that SMYD3 knockdown caused the upregulation of the cyclin-dependent kinase (CDK) inhibitors CDKN2A (p16INK4), CDKN2B (p15INK4B), CDKN3 and CDC25A, which may be responsible for the S phase arrest. In addition, the upregulation of CD40LG and downregulation of BIRC3 may explain the increased cell apoptosis rate after silencing SMYD3. We also discovered that SMYD3 bound on the promoter of CDKN2A and down-regulated its expression by triple-methylating H4K20. In addition, SMYD3 bound on the promoter of BIRC3 and up-regulated its expression by triple-methylating H3K4. Finally, knocking down SMYD3 could inhibit ovarian cancer growth in nude mice subcutaneous tumor model and PDX model.Conclusion: Our results demonstrated that SMYD3 was overexpressed in ovarian cancer and contributes to the regulation of tumor proliferation and apoptosis via SMYD3-H4K20me3-CDKN2A pathway and SMYD3-H3K4me3-BIRC3 pathway. Thus, SMYD3 is a promising epigenetic therapeutic target for ovarian cancer.

Signaling coupled epigenomic regulation of gene expression.

Inheritance of genomic information independent of the DNA sequence, the epigenetics, as well as gene transcription are profoundly shaped by serine/threonine and tyrosine signaling kinases and components of the chromatin remodeling complexes. To precisely respond to a changing external milieu, human cells efficiently translate upstream signals into post-translational modifications (PTMs) on histones and coregulators such as corepressors, coactivators, DNA-binding factors and PTM modifying enzymes. Because a protein with multiple residues for putative PTMs is expected to undergo more than one PTM in cells stimulated with growth factors, the outcome of combinational PTM codes on histones and coregulators is profoundly shaped by regulatory interplays between PTMs. The genomic functions of signaling kinases in cancer cells are manifested by the downstream effectors of cytoplasmic signaling cascades as well as translocation of the cytoplasmic signaling kinases to the nucleus. Signaling-mediated phosphorylation of histones serves as a regulatory switch for other PTMs, and connects chromatin remodeling complexes into gene transcription and gene activity. Here, we will discuss the recent advances in signaling-dependent epigenomic regulation of gene transcription using a few representative cancer-relevant serine/threonine and tyrosine kinases and their interplay with chromatin remodeling factors in cancer cells.

Extra-nuclear effects of estrogen on cortical bone in males require ERαAF-1.

Estradiol (E2) signaling via estrogen receptor alpha (ERα) is important for the male skeleton as demonstrated by ERα inactivation in both mice and man. ERα mediates estrogenic effects not only by translocating to the nucleus and affecting gene transcription but also by extra-nuclear actions e.g., triggering cytoplasmic signaling cascades. ERα contains various domains, and the role of activation function 1 (ERαAF-1) is known to be tissue specific. The aim of this study was to determine the importance of extra-nuclear estrogen effects for the skeleton in males and to determine the role of ERαAF-1 for mediating these effects. Five-month-old male wild-type (WT) and ERαAF-1-inactivated (ERαAF-10) mice were orchidectomized and treated with equimolar doses of 17β-estradiol (E2) or an estrogen dendrimer conjugate (EDC), which is incapable of entering the nucleus and thereby only initiates extra-nuclear ER actions or their corresponding vehicles for 3.5 weeks. As expected, E2 treatment increased cortical thickness and trabecular bone volume per total volume (BV/TV) in WT males. EDC treatment increased cortical thickness in WT males, whereas no effect was detected in trabecular bone. In ERαAF-10 males, E2 treatment increased cortical thickness, but did not affect trabecular bone. Interestingly, the effect of EDC on cortical bone was abolished in ERαAF-10 mice. In conclusion, extra-nuclear estrogen signaling affects cortical bone mass in males, and this effect is dependent on a functional ERαAF-1. Increased knowledge regarding estrogen signaling mechanisms in the regulation of the male skeleton may aid the development of new treatment options for male osteoporosis.

The Structural Basis of Ligand Perception and Signal Activation by Receptor Kinases.

Plants have evolved a family of unique membrane receptor kinases to orchestrate the growth and development of their cells, tissues, and organs. Receptor kinases also form the first line of defense of the plant immune system and allow plants to engage in symbiotic interactions. Here, we discuss recent advances in understanding, at the molecular level, how receptor kinases with lysin-motif or leucine-rich-repeat ectodomains have evolved to sense a broad spectrum of ligands. We summarize and compare the established receptor activation mechanisms for plant receptor kinases and dissect how ligand binding at the cell surface leads to activation of cytoplasmic signaling cascades. Our review highlights that one family of plant membrane receptors has diversified structurally to fulfill very different signaling tasks.

MYC and RAF: Key Effectors in Cellular Signaling and Major Drivers in Human Cancer.

The prototypes of the human MYC and RAF gene families are orthologs of animal proto-oncogenes that were originally identified as transduced alleles in the genomes of highly oncogenic retroviruses. MYC and RAF genes are now established as key regulatory elements in normal cellular physiology, but also as major cancer driver genes. Although the predominantly nuclear MYC proteins and the cytoplasmic RAF proteins have different biochemical functions, they are functionally linked in pivotal signaling cascades and circuits. The MYC protein is a transcription factor and together with its dimerization partner MAX holds a central position in a regulatory network of bHLH-LZ proteins. MYC regulates transcription conducted by all RNA polymerases and controls virtually the entire transcriptome. Fundamental cellular processes including distinct catabolic and anabolic branches of metabolism, cell cycle regulation, cell growth and proliferation, differentiation, stem cell regulation, and apoptosis are under MYC control. Deregulation of MYC expression by rearrangement or amplification of the MYC locus or by defects in kinase-mediated upstream signaling, accompanied by loss of apoptotic checkpoints, leads to tumorigenesis and is a hallmark of most human cancers. The critically controlled serine/threonine RAF kinases are central nodes of the cytoplasmic MAPK signaling cascade transducing converted extracellular signals to the nucleus for reshaping transcription factor controlled gene expression profiles. Specific mutations of RAF kinases, such as the prevalent BRAF(V600E) mutation in melanoma, or defects in upstream signaling or feedback loops cause decoupled kinase activities which lead to tumorigenesis. Different strategies for pharmacological interference with MYC- or RAF-induced tumorigenesis are being developed and several RAF kinase inhibitors are already in clinical use.

Retinal Axon Guidance Requires Integration of Eya and the Jak/Stat Pathway into Phosphotyrosine-Based Signaling Circuitries in Drosophila.

The transcriptional coactivator and phosphatase eyes absent (Eya) is dynamically compartmentalized between the nucleus and cytoplasm. Although the nuclear transcriptional circuits within which Eya operates have been extensively characterized, understanding of its cytoplasmic functions and interactions remains limited. Our previous work showed that phosphorylation of Drosophila Eya by the Abelson tyrosine kinase can recruit Eya to the cytoplasm and that eya-abelson interactions are required for photoreceptor axons to project to correct layers in the brain. Based on these observations, we postulated that photoreceptor axon targeting might provide a suitable context for identifying the cytoplasmic signaling cascades with which Eya interacts. Using a dose-sensitive eya misexpression background, we performed an RNA interference-based genetic screen to identify suppressors. Included among the top 10 hits were nonreceptor tyrosine kinases and multiple members of the Jak/Stat signaling network (hop, Stat92E, Socs36E, and Socs44A), a pathway not previously implicated in axon targeting. Individual loss-of-function phenotypes combined with analysis of axonal projections in Stat92E null clones confirmed the importance of photoreceptor autonomous Jak/Stat signaling. Experiments in cultured cells detected cytoplasmic complexes between Eya and Hop, Socs36E and Socs44A; the latter interaction required both the Src homology 2 motif in Socs44A and tyrosine phosphorylated Eya, suggesting direct binding and validating the premise of the screen. Taken together, our data provide new insight into the cytoplasmic phosphotyrosine signaling networks that operate during photoreceptor axon guidance and suggest specific points of interaction with Eya.

Structural Basis of Membrane Targeting by the Innate Immunity Adaptor TIRAP

Toll-like receptors (TLRs) are responsible for early immune system recognition and response to infection. Pathogen-activated TLRs trigger a cytoplasmic signaling cascade though adaptor proteins, starting with the TIR domain-containing adaptor protein (TIRAP). TIRAP contains a C-terminal TIR domain, which is responsible for association with TLRs and other adaptors including the myeloid differentiation primary response gene88 (MyD88) protein. Membrane recruitment of TIRAP is mediated by its N-terminal phosphoinositide (PIP)-binding motif (PBM). Upon ligand-mediated activation, TLRs are recruited to the PIP-enriched regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the membrane to activate the TLR signaling pathway. To understand the mechanism of TIRAP membrane targeting and the basis for its regulation, we functionally and structurally characterized TIRAP and its PBM using biophysical approaches. We show that TIRAP's PBM adopts a helical conformation in the presence of dodecylphosphocholine (DPC) micelles. NMR studies revealed that TIRAP PBM binds PIPs through its two conserved basic termini following a fast-exchange regime with moderate affinity. Mutation of these two basic regions abolishes PIP binding without distorting the helical structure of the peptide. Interestingly, association of TIRAP PBM to PIPs requires the acyl chains of the lipid. The solution NMR structure of TIRAP PBM exhibits a central relatively hydrophobic helix surrounded by the flexible N- and C-termini. Paramagnetic studies indicate that the helix is close to the micelle core, whereas the N- and C- termini are located on the micellar surface. Nuclear spin relaxation experiments indicate that this N- and C-termini of TIRAP PBM become more ordered when bound to PIPs. Thus, we propose that the central helix in PBM is responsible for membrane insertion, whereas the two sets of basic residues interact with PIPs to stabilize TIRAP's membrane interaction.

Dialing in EGFR Signaling

The ErbB family is a subfamily of receptor tyrosine kinases (RTKs). In RTKs, ligand binding at the extracellular region triggers diverse cytoplasmic signaling cascades. Exactly how ligand binding is translated into specific signaling outcomes remains incompletely understood. In this issue, Doerner etal. (2015) provide insights into a role that the juxtamembrane (JM) region of a representative ErbB kinase, EGFR, plays in this process.

Dissecting NF-κB signaling induced by genotoxic agents via genetic complementation of NEMO-deficient 1.3E2 cells.

The transcription factor NF-κB regulates expression of a diverse set of genes to modulate multiple biological and pathological processes. Among these, NF-κB activation in response to genotoxic agents has received considerable attention due to its role in regulating cancer cell resistance to chemo- and radiation therapy. Furthermore, induction of this pathway by endogenous damage is further implicated in normal developmental processes, such as B cell development, and premature aging, among others. This pathway also serves as a signaling model in which nuclear initiated signals (DNA damage) are communicated to a cytoplasmic target (IκB kinase and NF-κB). Several of the critical molecular events of this nuclear to cytoplasmic NF-κB signaling cascade were discovered, in part, by genetic complementation analyses of the NEMO-deficient 1.3E2 mouse pre-B cell line. This chapter describes methods used to generate and analyze such reconstitution cell systems and certain caveats that are critical for proper interpretation of NEMO mutant defects.

Structural Basis of Phosphoinositide (PIP) Recognition by the TIRAP PIP-Binding Motif

Toll-like receptors (TLRs) provide early immune system recognition and response to infection. TLRs activated by pathogens consequentially initiate a cytoplasmic signaling cascade though adaptor proteins, the first being the modular TIR domain-containing adaptor protein (TIRAP). TIRAP contains a C-terminal TIR domain, which is responsible for association with TLRs and other adaptors including the myeloid differentiation primary response gene88 (MyD88) protein. Membrane recruitment of TIRAP is mediated by its N-terminal PIP-binding motif (PBM). Upon ligand-mediated activation, TLRs are recruited to the PIP-enriched regions where TIRAP resides. At these sites, TIRAP recruits MyD88 to the membrane by bridging MyD88 to activate the TLR signaling pathway. To understand the mechanism of membrane targeting of TIRAP and the basis for its regulation, we functionally and structurally characterized its PBM using experimental and computational studies. TIRAP PBM adopts a folded conformation in membrane mimics, such as dodecylphosphocholine micelles, and binds PIPs. Structural rearrangements of TIRAP PBM were influenced by membrane interaction, with monodispersed PIPs inducing helical structure in the peptide. In contrast, monodispersed phosphatidylinositol and inositol trisphosphate did not promote structural changes in TIRAP PBM. NMR spectra reveal that TIRAP PBM binds PIPs in a fast exchange regime with a moderate affinity through two conserved basic regions. Solution NMR structure of TIRAP PBM shows a central short helix, and paramagnetic studies indicate that this region is close to the micelle core. Molecular dynamics simulations studies indicated that TIRAP PBM diffused to and interacted with a model membrane composed of palmitoyl oleoyl phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. Thus, we propose that two sets of basic residues contact both the head group and acyl chains of PIPs, whereas the central helix is responsible for membrane insertion.

Novel methylxanthine derivative-mediated anti-inflammatory effects in inflammatory bowel disease.

Family 18 chitinases have a binding capacity with chitin, a polymer of N-acetylglucosamine. Recent studies strongly suggested that chitinase 3-like 1 (CHI3L1, also known as YKL-40) and acidic mammalian chitinase, the two major members of family 18 chitinases, play a pivotal role in the pathogenesis of inflammatory bowel disease (IBD), bronchial asthma and several other inflammatory disorders. Based on the data from high-throughput screening, it has been found that three methylxanthine derivatives, caffeine, theophylline, and pentoxifylline, have competitive inhibitory effects against a fungal family 18 chitinase by specifically interacting with conserved tryptophans in the active site of this protein. Methylxanthine derivatives are also known as adenosine receptor antagonists, phosphodiesterase inhibitors and histone deacetylase inducers. Anti-inflammatory effects of methylxanthine derivatives have been well-documented in the literature. For example, a beneficial link between coffee or caffeine consumption and type 2 diabetes as well as liver cirrhosis has been reported. Furthermore, theophylline has a long history of being used as a bronchodilator in asthma therapy, and pentoxifylline has an immuno-modulating effect for peripheral vascular disease. However, it is still largely unknown whether these methylxanthine derivative-mediated anti-inflammatory effects are associated with the inhibition of CHI3L1-induced cytoplasmic signaling cascades in epithelial cells. In this review article we will examine the above possibility and summarize the biological significance of methylxanthine derivatives in intestinal epithelial cells. We hope that this study will provide a rationale for the development of methylxanthine derivatives, in particular caffeine, -based anti-inflammatory therapeutics in the field of IBD and IBD-associated carcinogenesis.

Molecular Mechanism for Plant Steroid Receptor Activation by Somatic Embryogenesis Co-Receptor Kinases

Brassinosteroids, which control plant growth and development, are sensed by the leucine-rich repeat (LRR) domain of the membrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1), but it is unknown how steroid binding at the cell surface activates the cytoplasmic kinase domain of the receptor. A family of somatic embryogenesis receptor kinases (SERKs) has been genetically implicated in mediating early brassinosteroid signaling events. We found a direct and steroid-dependent interaction between the BRI1 and SERK1 LRR domains by analysis of their complex crystal structure at 3.3 angstrom resolution. We show that the SERK1 LRR domain is involved in steroid sensing and, through receptor-co-receptor heteromerization, in the activation of the BRI1 signaling pathway. Our work reveals how known missense mutations in BRI1 and in SERKs modulate brassinosteroid signaling and the targeting mechanism of BRI1 receptor antagonists.

Target-based molecular signature characteristics of cervical adenocarcinoma and squamous cell carcinoma

There is an urgent need for molecular marker studies of adenocarcinoma (AC) and squamous cell carcinoma (SCC) of the uterine cervix. This study utilized oligomicroarray and pathway analyses to characterize a transcriptomic signature with molecular networks associated with AC and SCC. A 10K oligomicroarray was used to identify potential transcripts that were differentially expressed in cervical cancers from 28 patients and common reference RNAs from 17 different normal cervixes. Molecular networks were correlated using genomics tools to globally explore cellular pathways. Gene expression levels of 46 transcripts separated cancer samples into AC and SCC groups. Genes including: KRT17, IGFBP2, CALCA and VIPR1 were differentially expressed in AC and SCC. In addition, we identified a transcriptomic signature that predicted tumor classification and progression based upon its cellular processes. The downregulated signatures for SCC were cell death of pheochromocytoma cells (P=0.0037), apoptosis of neurons (P=0.009) and damage to DNA (P=0.0038). By contrast, the upregulated molecular signatures in AC were immunological disorder (P=0.006), splenomegaly (P=0.0053) and hepatic system disorder (P=0.006). The G2/M DNA damage checkpoint regulation pathway (P=0.05) was found to be significantly linked to IGF1R as a new regulatory component of a putative cytoplasmic signaling cascade in SCC. By contrast, the antigen presenting canonical pathway (P=0.038) appeared to be linked to PPARγ in AC. Taken together, these experiments provide important new information regarding the role of molecular networks in mediating SCC and AC, possibly through two independent pathways, and contribute to provide new targets for the prevention and treatment of cervical cancer.

Overexpressed A20 in Refractory/Relapse B-ALL May Serve As a Potential Therapeutic Target

Abstract 4816Treatment failure of precursor B-lymphoblastic leukemia (B-ALL), also known as B-cell acute lymphoblastic leukemia and B-cell acute lymphocytic leukemia, remains a formidable problem. The molecular abnormalities responsible for relapse or drug resistance are still not clear. NF-κB-dependent gene expression and apoptosis plays crucial roles in numerous cellular processes, and defects in their regulation may contribute to a variety of diseases like cancers. As a key regulatory component of the cytoplasmic signaling cascade that mediates NF-κB activation in response to DNA damage, the zinc finger-containing protein A20 (also known as TNF-α-induced protein 3, TNFAIP3) has been found to correlate with drug resistance of breast cancer, prostate cancer and acute myeloid leukemia. To investigate the function of A20 in the development of B-ALL, we analyzed the expression levels of A20 and NF-κB genes in peripheral blood mononuclear cells (PBMCs) from patients (aged 3–72 years, median 30.5) with newly diagnosed B-ALL, B-ALL in complete remission (CR), and refractory/relapse B-ALL. Twenty-nine healthy individuals (aged 20–57, median 28) served as controls. We found that the expression levels of A20 in newly diagnosed B-ALL patients (23.450±16.164) and refractory/relapse B-ALL patients (26.108±12.301) were significantly higher than those from healthy controls (9.865±5.370) (P<0.0001, P<0.0001), while the expression levels of A20 in B-ALL CR patients (2.826±2.415) were significantly decreased and was lower than those from healthy controls (P=0.008). The expression levels of NF-κB in newly diagnosed B-ALL patients (2.025±0.986) were significantly higher than healthy controls (0.337±0.311) (P<0.0001), while the expression levels of NF-κB in refractory/relapse B-ALL patients (0.668±0.707) and B-ALL CR patients (0.405±0.172) were similar to healthy group (P=0.057, P=0.559). The expression levels of A20 had a positive correlation with the expression levels of NF-κB in de novo B-ALL (rs=0.7473, P=0.021), however, the expression levels of A20 in refractory/relapse B-ALL and B-ALL CR group did not show significantly correlation with the expression levels of NF-κB. In conclusion, overexpression of A20 is a feature in de novo and refractory/relapse B-ALL, which may loss their negative regulating function for NF-κB. The expression level of A20 may correlate with the clinical stages of B-ALL, indicating A20 might be considered as a prognostic marker of B-ALL and a potential therapeutic target for refractory/relapse B-ALL. Disclosures:Xu:National Natural Science Foundation of China (No. 91129720): Research Funding. Wu:National Natural Science Foundation of China (No. 91129720): Research Funding. Zhang:National Natural Science Foundation of China (No. 91129720): Research Funding. Liu:National Natural Science Foundation of China (No. 91129720): Research Funding. Chen:National Natural Science Foundation of China (No. 91129720): Research Funding. Yang:National Natural Science Foundation of China (No. 91129720): Research Funding. Li:National Natural Science Foundation of China (No. 91129720): Research Funding. Li:National Natural Science Foundation of China (No. 91129720): Research Funding.

Activation of rapid oestrogen signalling in aggressive human breast cancers

Oestrogen receptors can mediate rapid activation of cytoplasmic signalling cascades by recruiting Src and PI3K. However, the involvement of this pathway in breast cancer remains poorly defined. We have previously shown that methylation of ERα is required for the formation of the ERα/Src/PI3K complex and that ERα is hypermethylated in a subset of breast cancers. Here, we used Proximity Ligation Assay to demonstrate that this complex is present in the cytoplasm of breast cancer cell lines as well as formalin-fixed, paraffin-embedded tumours. Of particular interest, the analysis of 175 breast tumours showed that overexpression of this complex in a subset of breast tumours correlates to the activation of the downstream effector Akt. Survival analysis revealed that high expression of this complex is an independent marker of poor prognosis and associated with reduced disease-free survival. Our data introduces the new concept that the rapid oestrogen pathway is operative in vivo. It also provides a rationale for patient stratification defined by the activation of this pathway and the identification of target therapies.

Mechanical Regulation of Nuclear Structure and Function

Mechanical loading induces both nuclear distortion and alterations in gene expression in a variety of cell types. Mechanotransduction is the process by which extracellular mechanical forces can activate a number of well-studied cytoplasmic signaling cascades. Inevitably, such signals are transduced to the nucleus and induce transcription factor-mediated changes in gene expression. However, gene expression also can be regulated through alterations in nuclear architecture, providing direct control of genome function. One putative transduction mechanism for this phenomenon involves alterations in nuclear architecture that result from the mechanical perturbation of the cell. This perturbation is associated with direct mechanical strain or osmotic stress, which is transferred to the nucleus. This review describes the current state of knowledge relating the nuclear architecture and the transfer of mechanical forces to the nucleus mediated by the cytoskeleton, the nucleoskeleton, and the LINC (linker of the nucleoskeleton and cytoskeleton) complex. Moreover, remodeling of the nucleus induces alterations in nuclear stiffness, which may be associated with cell differentiation. These phenomena are discussed in relation to the potential influence of nuclear architecture-mediated mechanoregulation of transcription and cell fate.

Gαo potentiates estrogen receptor α activity via the ERK signaling pathway

The estrogen receptor α (ERα) is a transcription factor that mediates the biological effects of 17β-estradiol (E(2)). ERα transcriptional activity is also regulated by cytoplasmic signaling cascades. Here, several Gα protein subunits were tested for their ability to regulate ERα activity. Reporter assays revealed that overexpression of a constitutively active Gα(o) protein subunit potentiated ERα activity in the absence and presence of E(2). Transient transfection of the human breast cancer cell line MCF-7 showed that Gα(o) augments the transcription of several ERα-regulated genes. Western blots of HEK293T cells transfected with ER±Gα(o) revealed that Gα(o) stimulated phosphorylation of ERK 1/2 and subsequently increased the phosphorylation of ERα on serine 118. In summary, our results show that Gα(o), through activation of the MAPK pathway, plays a role in the regulation of ERα activity.