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Introduction: The independent diagnostic value of inflammatory markers neutrophil to lymphocyte ratio (NLR) and platelet to lymphocyte ratio (PLR) and the diagnostic efficacy of NLR, derived neutrophil to lymphocyte ratio (dNLR), PLR, and lymphocyte-to-monocyte ratio (LMR) in glioma cases remain unclear. We investigated the correlation of preoperative peripheral blood inflammatory markers with pathological grade, Ki-67 Proliferation Index, and IDH-1 gene phenotype in patients with glioma, focusing on tumor grade and prognosis. Methods: We retrospectively analyzed the clinical, pathological, and laboratory data of 334 patients with glioma with varying grades and 345 with World Health Organization (WHO I) meningioma who underwent initial surgery at the Affiliated Hospital of Jining Medical University from December 2019 to December 2021. The diagnostic value of peripheral blood inflammatory markers for glioma was investigated. Results: The proportion of men smoking and drinking was significantly higher in the glioma group than in the meningioma group ( P .05). LMR, NLR, dNLR, and PLR, varied significantly among different glioma types ( P

Abstract Metastasis is the leading cause of cancer-related deaths. Transforming growth factor beta (TGF-β) signaling drives metastasis and is strongly enhanced during cancer progression. Yet, the use of on-target TGF-β signaling inhibitors in the treatment of cancer patients remains unsuccessful, highlighting a gap in the understanding of TGF-β biology that limits the establishment of efficient anti-metastatic therapies. Here, we show that TGF-β signaling hyperactivation in breast cancer cells is required for metastasis and relies on increased small extracellular vesicle (sEV) secretion. Demonstrating sEV’s unique role, TGF-β signaling levels induced by sEVs exceed the activity of matching concentrations of soluble ligand TGF-β. Further, genetic disruption of sEV secretion in highly-metastatic breast cancer cells impairs cancer cell aggressiveness by reducing TGF-β signaling to nearly-normal levels. Otherwise, TGF-β signaling activity in non-invasive breast cancer cells is inherently low, but can be amplified by sEVs, enabling invasion and metastasis of poorly-metastatic breast cancer cells. Underscoring the translational potential of inhibiting sEV trafficking in advanced breast cancers, treatment with dimethyl amiloride (DMA) decreases sEV secretion, TGF-β signaling activity, and breast cancer progression in vivo. Targeting both the sEV trafficking and TGF-β signaling by combining DMA and SB431542 at suboptimal doses potentiated this effect, normalizing the TGF-β signaling in primary tumors to potently reduce circulating tumor cells, metastasis, and tumor self-seeding. Collectively, this study establishes sEVs as critical elements in TGF-β biology, demonstrating the feasibility of inhibiting sEV trafficking as a new therapeutic approach to impair metastasis by normalizing TGF-β signaling levels in breast cancer cells.

Background: Metastasis is the main cause of cancer-related deaths, but efficient targeted therapies against metastasis are still missing. Major gaps exist in our understanding of the metastatic cascade, as existing methods cannot combine sensitivity, robustness, and practicality to dissect cancer progression. Addressing this issue requires improved strategies to distinguish early metastatic colonization from metastatic outgrowth. Methods: Luciferase-labelled MDA-MB-231, MCF7, and 4T1 breast cancer cells were spiked into samples from tumour-naïve mice to establish the limit of detection for disseminated tumour cells. Luciferase-labelled breast cancer cells (±unlabelled cancer-associated fibroblasts; CAFs) were orthotopically implanted in immunocompromised mice. An ex vivo luciferase assay was used to quantify tumour cell dissemination. Results: In vitro luciferase assay confirmed a linear and positive correlation between cancer cell numbers and the bioluminescence detected at single cell level in blood, brain, lung, liver, and mammary fat pad samples. Remarkably, single luciferase-labelled cancer cells were detectable in all of these sites, as the bioluminescence quantified in the analysed samples was substantially higher than background levels. Ex vivo, circulating tumour cells, metastasis, and tumour self-seeding were detected in all samples from animals implanted with highly metastatic luciferase-labelled MDA-MB-231 cells. In turn, detection of poorly metastatic luciferase-labelled MCF7 cells was scarce but significantly enhanced upon co-implantation with CAFs as early as 20 days after the experiment was initiated. Conclusions: These results demonstrate the feasibility of using an ultrasensitive luciferase-based method to dissect the mechanisms of early metastatic colonization to improving the development of antimetastatic therapies.

Background: Glioblastoma is characterised by extensive infiltration into the brain parenchyma, leading to inevitable tumor recurrence and therapeutic failure. Future treatments will need to target the specific biology of tumour recurrence, but our current understanding of the underlying mechanisms is limited. Significantly, there is a lack of available methods and models that are tailored to the examination of tumour recurrence. Methods: NOD-SCID mice were orthotopically implanted with luciferase-labelled donor U87MG or MU20 glioblastoma cells. Four days later, an unlabelled recipient tumor was implanted on the contralateral side. The mice were euthanised at a humane end-point and tissue and blood samples were collected for ex vivo analyses. Results: The ex vivo analyses of the firefly-labelled MU20 tumours displayed extensive invasion at the primary tumour margins, whereas the firefly-labelled U87MG tumours exhibited expansive phenotypes with no evident invasions at the tumour margins. Luciferase signals were detected in the contralateral unlabelled recipient tumours for both the U87MG and MU20 tumours compared to the non-implanted control brain. Remarkably, tumour cells were uniformly detected in all tissue samples of the supratentorial brain region compared to the control tissue, with single tumour cells detected in some tissue samples. Circulating tumour cells were also detected in the blood samples of most of the xenografted mice. Moreover, tumour cells were detected in the lungs of all of the mice, a probable event related to haematogenous dissemination. Similar results were obtained when the U87MG cells were alternatively labelled with gaussian luciferase. Conclusions: These findings describe a systemic disease model for glioblastoma which can be used to investigate recurrence biology and therapeutic efficacy towards recurrence.

Epithelial-mesenchymal transition (EMT) is crucial to metastasis by increasing cancer cell migration and invasion. At the cellular level, EMT-related morphological and functional changes are well established. At the molecular level, critical signaling pathways able to drive EMT have been described. Yet, the translation of EMT into efficient diagnostic methods and anti-metastatic therapies is still missing. This highlights a gap in our understanding of the precise mechanisms governing EMT. Here, we discuss evidence suggesting that overcoming this limitation requires the integration of multiple omics, a hitherto neglected strategy in the EMT field. More specifically, this work summarizes results that were independently obtained through epigenomics/transcriptomics while comprehensively reviewing the achievements of proteomics in cancer research. Additionally, we prospect gains to be obtained by applying spatio-temporal multiomics in the investigation of EMT-driven metastasis. Along with the development of more sensitive technologies, the integration of currently available omics, and a look at dynamic alterations that regulate EMT at the subcellular level will lead to a deeper understanding of this process. Further, considering the significance of EMT to cancer progression, this integrative strategy may enable the development of new and improved biomarkers and therapeutics capable of increasing the survival and quality of life of cancer patients.

Abstract To identify novel cancer therapies, the tumor microenvironment (TME) has received a lot of attention in recent years in particular with the advent of clinical successes achieved by targeting immune checkpoint inhibitors (ICIs). The TME consists of multiple cell types that are embedded in the extracellular matrix (ECM), including immune cells, endothelial cells and cancer associated fibroblasts (CAFs), which communicate with cancer cells and each other during tumor progression. CAFs are a dominant and heterogeneous cell type within the TME with a pivotal role in controlling cancer cell invasion and metastasis, immune evasion, angiogenesis and chemotherapy resistance. CAFs mediate their effects in part by remodeling the ECM and by secreting soluble factors and extracellular vesicles. Exosomes are a subtype of extracellular vesicles (EVs), which contain various biomolecules such as nucleic acids, lipids, and proteins. The biomolecules in exosomes can be transmitted from one to another cell, and thereby affect the behavior of the receiving cell. As exosomes are also present in circulation, their contents can also be explored as biomarkers for the diagnosis and prognosis of cancer patients. In this review, we concentrate on the role of CAFs-derived exosomes in the communication between CAFs and cancer cells and other cells of the TME. First, we introduce the multiple roles of CAFs in tumorigenesis. Thereafter, we discuss the ways CAFs communicate with cancer cells and interplay with other cells of the TME, and focus in particular on the role of exosomes. Then, we elaborate on the mechanisms by which CAFs-derived exosomes contribute to cancer progression, as well as and the clinical impact of exosomes. We conclude by discussing aspects of exosomes that deserve further investigation, including emerging insights into making treatment with immune checkpoint inhibitor blockade more efficient.

Testicular germ cell tumours (TGCTs) are the most common malignancy in young men. Originating from foetal testicular germ cells that fail to differentiate correctly, TGCTs appear after puberty as germ cell neoplasia in situ cells that transform through unknown mechanisms into distinct seminoma and non-seminoma tumour types. A balance between activin and BMP signalling may influence TGCT emergence and progression, and we investigated this using human cell line models of seminoma (TCam-2) and non-seminoma (NT2/D1). Activin A- and BMP4-regulated transcripts measured at 6 h post-treatment by RNA-sequencing revealed fewer altered transcripts in TCam-2 cells but a greater responsiveness to activin A, while BMP4 altered more transcripts in NT2/D1 cells. Activin significantly elevated transcripts linked to pluripotency, cancer, TGF-β, Notch, p53, and Hippo signalling in both lines, whereas BMP4 altered TGF-β, pluripotency, Hippo and Wnt signalling components. Dose-dependent antagonism of BMP4 signalling by activin A in TCam-2 cells demonstrated signalling crosstalk between these two TGF-β superfamily arms. Levels of the nuclear transport protein, IPO5, implicated in BMP4 and WNT signalling, are highly regulated in the foetal mouse germline. IPO5 knockdown in TCam-2 cells using siRNA blunted BMP4-induced transcript changes, indicating that IPO5 levels could determine TGF-β signalling pathway outcomes in TGCTs.

Metastasis is the leading cause of death for cancer patients. During cancer progression, the initial detachment of cells from the primary tumor and the later colonization of a secondary organ are characterized as limiting steps for metastasis. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) are opposite dynamic multistep processes that enable these critical events in metastasis by altering the phenotype of cancer cells and improving their ability to migrate, invade and seed at distant organs. Among the molecular pathways that promote tumorigenesis in late-stage cancers, transforming growth factor-β (TGF-β) is described as an EMT master inducer by controlling different genes and proteins related to cytoskeleton assembly, cell-cell attachment and extracellular matrix remodeling. Still, despite the successful outcomes of different TGF-β pharmacological inhibitors in cell culture (in vitro) and animal models (in vivo), results in cancer clinical trials are poor or inconsistent at least, highlighting the existence of crucial components in human cancers that have not been properly explored. Here we review most recent findings to provide perspectives bridging the gap between on-target anti-TGF-β therapies in vitro and in pre-clinical models and the poor clinical outcomes in treating cancer patients. Specifically, we focus on (i) the dual roles of TGF-β signaling in cancer metastasis; (ii) dynamic signaling; (iii) functional differences of TGF-β free in solution vs. in exosomes; (iv) the regulatory effects of tumor microenvironment (TME) – particularly by cancer-associated fibroblasts – on TGF-β signaling pathway. Clearly identifying and establishing those missing links may provide strategies to revitalize and clinically improve the efficacy of TGF-β targeted therapies.

ABSTRACT: Nanogeology is a subject that is a combination of geology and nanoscale science, and it has been a frontier field in recent years. It is also a new subject with the features of intersectionality and multidisciplinary. Digging deeper into geological problems and nanoscale phenomena helps better revealing the more essential mechanisms and processes in geological science, which is also an evitable path in the development of geology. In this paper, we elaborate the concept, feature and main subdisciplines, and summarize three stages of nanogeology development from preliminary research in the 1990s to subject formation in China. After summarizing the researchers’ achievements in this field, we illustrate some primary research progresses of nanogeology in China as eight subdisciplines. On the basis of the above content, we propose the development prospect of nanogeology in China. There are many geologic problems with scientific values and economic benefits, such as research of geologic fundamental problems, resource exploration and development, mechanism study and prediction of geological activities (disasters), mechanism research and management of environmental pollution and others. Nanogeology has a great potential in China to solve all of these problems. As a result, the theories and methods of nanogeology will become enriching and advanced. It offers important theoretical basis and technological methods to deal with major issues concerning the national economy and the people’s livelihoods, such as the prediction of geological activities, as well as resource distribution and its exploration and utilization.

Abstract Background Transformation by oncogene Ras overcomes TGF-β mediated growth inhibition in epithelial cells. However, it cooperates with each other to mediate epithelial to mesenchymal transition (EMT). The mechanism of how these two pathways interact with each other is controversial. Methods Molecular techniques were used to engineer expression plasmids for Ras, SPRY, TGF-β receptors, type I and II and ubiquitin. Immunoprecipitation and western blots were employed to determine protein-protein interactions, preotein levels, protein phosphorylation while immunofluorecesent staining for molecular co-localization. TGF-β signalling activities is also determined by its luciferase reporter assay. Trans-well assays were used to measure cell migration and invasion. Results Ras interacts with the SPSB1’s SPRY domain to enhance TGF-β signaling. Ras interacts and colocalizes with the TGF-β type II receptor’s (TβRII) negative regulator SPSB1 on the cell membrane, consequently promoting SPSB1 protein degradation via enhanced mono- and di-ubiquitination. Reduced SPSB1 levels result in the stablization of TβRII, in turn the increase of receptor levels significantly enhance Smad2/3 phosphorylation and signaling. Importantly, forced expression of SPSB1 in Ras transformed cells suppresses TGF-β signaling and its mediated migration and invasion. Conclusion Ras positively cooperates with TGF-β signaling by reducing the cellular protein levels of TβRII negative regualtor SPSB1.

Abstract Background C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined. Methods We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells. Results Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain. Conclusions SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells.

In order to ensure the stability and high accuracy on price forecasting, a novel idea is found that magnitude differences in the original data have an effect on price forecasting based on experiments. And a general data pre-processing method for the original data magnitude is put forward in this paper according to the novel idea. The new data pre-processing method that normalizes the magnitude of the original data is described in detail in this paper. The different data and models are used to verify the new idea and the generality of the proposed method. And the discrete data are the data of CNY exchange rate and agricultural product price. The different models are improved models of Back Propagation (BP) neural network and Radial Basis Function (RBF) neural network. Compared to other magnitudes, normalizing the magnitude of the original data to a smaller magnitude obtains the best result. And in this paper the smaller magnitude is the order 1. The forecasting average accuracy of the experiments done with the data of CNY exchange rate based on BP neural network and RBF neural network gets 99.14 percent and 99.27 percent. The forecasting average accuracy of the experiments completed with the data of agriculture product price based on BP neural network and RBF neural network gets 98.54 percent and 97.81 percent. Experiment demonstrates that the data pre-processing method has the great generality on discrete data and models. The data pre-processing method proposed in this paper reduces the differences between data which affect the accuracy of the prediction models to a degree. The generality of the proposed method is proved that the innovative idea and method are meaningful and useful for the price forecasting.

The natural cracking of crude oils in deep reservoirs has gained great interest due to continuously increasing depth of petroleum exploration and exploitation. Complex oil compositions and surroundings as well as complicated geological evolutions make oil cracking in nature much more complex than industrial pyrolysis. So far, numerous studies, focused on this topic, have made considerable progress although there still exist some drawbacks. However, a comprehensive review on crude oil cracking is yet to be conducted. This article systematically reviews the controlling factors of oil cracking from six aspects, namely, oil compositions, temperature and time, pressure, water, minerals and solid organic matter. We compare previous experimental and modelling results and present new field cases. In the following, we evaluate the prevailing estimation methods for the extent of oil cracking, and elucidate other factors that may interfere with the application of these estimation methods. This review will be helpful for further investigations of crude oil cracking and provides a guide for estimation of the cracking extent of crude oils. [ABSTRACT FROM AUTHOR]

Phomopsis vexans, which causes Phomopsis blight of eggplant, has been reported worldwide. To study the biocontrol of this disease, 162 leaf and fruit samples of eggplant Phomopsis blight were collected from Hunan, Hubei, Jiangxi, Sichuan, Zhejiang, Fujian, Guangdong and Anhui Provinces from 2017 to 2019. Eighty-seven pathogenic fungus isolates were identified as P. vexans. The following studies were conducted: screening of sporulation medium, spore morphology analysis, mycovirus detection and identification of novel mycoviruses in these isolates. The results showed that eggplant tissue medium was the most suitable medium for rapid sporulation, and all isolates had mycoviruses consisting of mainly mixed infections. The genome of these mycoviruses varied from 1-15 kb. Five novel mycoviruses infecting P. vexans were obtained, including "Phomopsis vexans fusarivirus 1" (PvFV1), "Phomopsis vexans ourmia-like virus 1" (PvOLV1), "Phomopsis vexans endornavirus 2" (PvEV2), "Phomopsis vexans partitivirus 1" (PvPV1) and "Phomopsis vexans victorivirus L1" (PvVVL1). Thus, PvVVL1 displays a unique genome structure, and this is the first report of a victorivirus consisting of two segments and of a deltapartitivirus infecting the fungus host. [ABSTRACT FROM AUTHOR]

Current cancer therapy such as chemotherapy and radiography are known to possess many side effects that could lead to serious complications. The application of natural product as complementary and alternative treatment provides significant advantages. It could increase the sensitivity of chemotherapy and radiography while at the same time reducing their associated side effects and complications. With regards to anticancer drugs, more than 50% of the modern oncological drugs are derived from natural products. An edible mushroom, Ganoderma lucidum has long been used in traditional and conventional medicine in China, for the prevention and treatment of various human diseases. The fungus possesses a wide variety of bioactive compounds present in its fruiting bodies, mycelium and spores, including anticancer property that has been proven in in vitro and in vivo studies. The carcinostatic effects of G. lucidum have been shown in a variety of cancer cell lines, including breast, pancreas, lung, colon, skin and prostate. This paper presents in vitro and in vivo studies of the anticancer properties of G. lucidum in cancers. [ABSTRACT FROM AUTHOR]

Def6 protein has been highly associated with autoimmune diseases and become one of risk gene in systemic lupus erythematosus (SLE) disease. Def6 deficiency is spontaneously affecting the development of systemic autoimmune disorder in mice as well as in human. Def6 or IRF-4- binding proteins (IBP) that mostly found among lymph node and thymus are associated with cell survival and cell proliferation. Def6 protein as a novel biomarker has many important functions and roles in human immune system. Def6 is believed to be a potential therapeutic pharmacological target or/and potential drug for some malignancy diseases and pathological disorders. Current studies reveal Def6 expression and regulations not only are found in autoimmune diseases but also in human cancer and chronic inflammatory diseases. High expression of Def6 is correlated with invasive tumours and malignant stage of various cancers. This review article sums up Def6 expression and regulation in some diseases stated above. Collectively, this review article provides novel studies concerning Def6 as a protein, its correlation with other genes, its mechanism and its expression in some diseases, particularly in cancer, autoimmune diseases and chronic inflammatory diseases. [ABSTRACT FROM AUTHOR]

Background: C-terminal Src kinase (Csk) and Csk-homologous kinase (Chk) are the major endogenous inhibitors of Src-family kinases (SFKs). They employ two mechanisms to inhibit SFKs. First, they phosphorylate the C-terminal tail tyrosine which stabilizes SFKs in a closed inactive conformation by engaging the SH2 domain in cis. Second, they employ a non-catalytic inhibitory mechanism involving direct binding of Csk and Chk to the active forms of SFKs that is independent of phosphorylation of their C-terminal tail. Csk and Chk are co-expressed in many cell types. Contributions of the two mechanisms towards the inhibitory activity of Csk and Chk are not fully clear. Furthermore, the determinants in Csk and Chk governing their inhibition of SFKs by the non-catalytic inhibitory mechanism are yet to be defined. Methods: We determined the contributions of the two mechanisms towards the inhibitory activity of Csk and Chk both in vitro and in transduced colorectal cancer cells. Specifically, we assayed the catalytic activities of Csk and Chk in phosphorylating a specific peptide substrate and a recombinant SFK member Src. We employed surface plasmon resonance spectroscopy to measure the kinetic parameters of binding of Csk, Chk and their mutants to a constitutively active mutant of the SFK member Hck. Finally, we determined the effects of expression of recombinant Chk on anchorage-independent growth and SFK catalytic activity in Chk-deficient colorectal cancer cells. Results: Our results revealed Csk as a robust enzyme catalysing phosphorylation of the C-terminal tail tyrosine of SFKs but a weak non-catalytic inhibitor of SFKs. In contrast, Chk is a poor catalyst of SFK tail phosphorylation but binds SFKs with high affinity, enabling it to efficiently inhibit SFKs with the non-catalytic inhibitory mechanism both in vitro and in transduced colorectal cancer cells. Further analyses mapped some of the determinants governing this non-catalytic inhibitory mechanism of Chk to its kinase domain. Conclusions: SFKs are activated by different upstream signals to adopt multiple active conformations in cells. SFKs adopting these conformations can effectively be constrained by the two complementary inhibitory mechanisms of Csk and Chk. Furthermore, the lack of this non-catalytic inhibitory mechanism accounts for SFK overactivation in the Chk-deficient colorectal cancer cells. [ABSTRACT FROM AUTHOR]

Ustilaginoidea virens, the causal agent of rice false smut, is one of the most devastating grain diseases that causes loss of yield in most rice-growing areas worldwide. In this study, we performed a dsRNA screen to isolate mycoviruses from 35 U. virens strains. The results revealed that 34 of the tested isolates were infected by various dsRNA elements, displaying highly viral diversity and mixed infections. We characterized a 5.3 kbp dsRNA from a typical isolate containing dsRNA segments with sizes ranging from 0.5 to 5.3 kbp. Sequence analysis of its genomic properties indicated that it is a novel victorivirus, named Ustilaginoidea virens RNA virus 5 (UvRV5), that belongs to the family Totiviridae. RT-PCR detection was performed and indicated that not all the dsRNA bands that were 5.3 kbp in size contained UvRV5. Moreover, the genetic relatedness of all the U. virens strains was estimated according to phylogenetic analysis of the partial intergenic spacer region (IGS) sequences. However, concordance was not found between the dsRNA profiles and the IGS-based genetic relatedness of their host fungi. [ABSTRACT FROM AUTHOR]

Background: Transforming growth factor β (TGF-β) signalling regulates the development of embryos and tissue homeostasis in adults. In conjunction with other oncogenic changes, long-term perturbation of TGF-β signalling is associated with cancer metastasis. Although TGF-β signalling can be complex, many of the signalling components are well defined, so it is possible to develop mathematical models of TGF-β signalling using reduction and scaling methods. The parameterization of our TGF-β signalling model is consistent with experimental data. Results: We developed our mathematical model for the TGF-β signalling pathway, i.e. the RF-model of TGF-β signalling, using the "rapid equilibrium assumption" to reduce the network of TGF-β signalling reactions based on the time scales of the individual reactions. By adding time-delayed positive feedback to the inherent time-delayed negative feedback for TGF-β signalling. We were able to simulate the sigmoidal, switch-like behaviour observed for the concentration dependence of long-term (> 3 hours) TGF-β stimulation. Computer simulations revealed the vital role of the coupling of the positive and negative feedback loops on the regulation of the TGF-β signalling system. The incorporation of time-delays for the negative feedback loop improved the accuracy, stability and robustness of the model. This model reproduces both the short-term and long-term switching responses for the intracellular signalling pathways at different TGF-β concentrations. We have tested the model against experimental data from MEF (mouse embryonic fibroblasts) WT, SV40-immortalized MEFs and Gp130F/F MEFs. The predictions from the RF-model are consistent with the experimental data. Conclusions: Signalling feedback loops are required to model TGF-β signal transduction and its effects on normal and cancer cells. We focus on the effects of time-delayed feedback loops and their coupling to ligand stimulation in this system. The model was simplified and reduced to its key components using standard methods and the rapid equilibrium assumption. We detected differences in short-term and long-term signal switching. The results from the RF-model compare well with experimental data and predict the dynamics of TGF-β signalling in cancer cells with different mutations. [ABSTRACT FROM AUTHOR]

Cell-cell communication is critical across an assortment of physiological and pathological processes. Extracellular vesicles (EVs) represent an integral facet of intercellular communication largely through the transfer of functional cargo such as proteins, messenger RNAs (mRNAs), microRNA (miRNAs), DNAs and lipids. EVs, especially exosomes and shed microvesicles, represent an important delivery medium in the tumour micro-environment through the reciprocal dissemination of signals between cancer and resident stromal cells to facilitate tumorigenesis and metastasis. An important step of the metastatic cascade is the reprogramming of cancer cells from an epithelial to mesenchymal phenotype (epithelial-mesenchymal transition, EMT), which is associated with increased aggressiveness, invasiveness and metastatic potential. There is now increasing evidence demonstrating that EVs released by cells undergoing EMT are reprogrammed (protein and RNA content) during this process. This review summarises current knowledge of EV-mediated functional transfer of proteins and RNA species (mRNA, miRNA, long non-coding RNA) between cells in cancer biology and the EMT process. An in-depth understanding of EVs associated with EMT, with emphasis on molecular composition (proteins and RNA species), will provide fundamental insights into cancer biology. [ABSTRACT FROM AUTHOR]

A novel double-stranded RNA (dsRNA) mycovirus, consisting of three dsRNA genome segments and possibly belonging to the family Chrysoviridae, was isolated from the filamentous phytopathogenic fungus Colletotrichum gloeosporioides and designated as Colletotrichum gloeosprioides chrysovirus 1 (CgCV1). The three dsRNAs of the CgCV1 genome with lengths of 3397, 2869, and 2630 bp (dsRNAs1-3) were found to contain a single open reading frame (ORF) putatively encoding the RNA-dependent RNA polymerase (RdRp), a capsid protein, and a protease, respectively, all of which exhibited some degree of sequence similarity to the comparable putative proteins encoded by the genus Chrysovirus. The 5' - and 3'-untranslated regions in each dsRNA segment contained similar sequences that were strictly conserved at the termini. Moreover, isometric virus-like particles (VLPs) with a diameter of approximately 40 nm were extracted from fungal mycelia. Phylogenetic analysis based on the conserved dsRNA1-encoded RdRp showed that CgCV1 is a new virus belonging to the Chrysoviridae family. BLAST analysis revealed the presence of CgCV1-like sequences in the chromosomes of Medicago truncatula and Solanum tuberosum. Moreover, some sequences in the transcriptome shotgun assembly (TSA) library and expressed sequence tag database (ESTdb) of other eudicot and monocot plants were also found to be related to CgCV1. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the clinical efficacy and safety of low-dose 90Sr-90Y therapy combined with the topical application of 0.5% timolol maleate solution for the treatment of superficial infantile hemangiomas (IHs). A total of 72 infants with hemangiomas were allocated at random into the observation group (17 cases aged ≤3 months, 20 cases aged >3 months) or the control group (15 cases aged ≤3 months, 20 cases aged >3 months). The observation group was treated with low-dose 90Sr-90Y combined with timolol, while the control group received an identical dose of 90Sr-90Y with physiological saline. Data were collected for statistical analysis, and treatment efficacy was compared between the two groups. In the observation group, 100% (37/37) of subjects exhibited an 'excellent' response to the treatment, while 94.1% (16/17) of patients aged ≤3 months and 85.0% (17/20) aged >3 months were classed as being cured. In the control group, the treatment was classed as 'effective' in 100% (35/35) of the subjects, while the excellent response rate was 86.7% (13/15) among the infants aged ≤3 months and 75.0% (15/20) among the infants aged >3 months. The 'cure' rates in the control group were 66.7% (10/15) and 60.0% (12/20) for the ≤3-month- and >3-month-old subjects, respectively. The excellent response and cure rates were notably higher in the observation group than those in the control group. Comparison between the two groups revealed a χ² value of 13.90 (P<0.01) for excellent responses in subjects aged ≤3 months, while for patients aged >3 months the χ² value was 28.57 (P<0.01). Analysis of the cure responses gave similar results [≤3 months, χ²=23.22 (P<0.01); >3 months, χ²=15.67 (P<0.01)]. At 3-4 months after the first course of treatment, the cure rate was 33.3% (11/33) in the observation group, which was significantly higher than the rate of 18.32% (4/22) in the control group (χ²=5.92, P<0.05). No serious adverse reactions were observed in either group. In summary, low-dose 90Sr-90Y therapy combined with the topical application of 0.5% timolol maleate induces a rapid response in superficial IH, with excellent efficacy and no obvious adverse reactions, and may represent a clinically applicable intervention. [ABSTRACT FROM AUTHOR]

Background: Metastasis is a process where only a small subset of cells is capable of successfully migrating to and propagating at secondary sites. TGF-β signalling is widely known for its role in cancer metastasis and is associated with cell migration in whole cell populations. Findings: We extend these findings by investigating the role of TGF-β signalling in promoting migration and motility by imaging the signalling activity in live, individual MDA-MB-231 cancer cells utilizing a novel Smad3 Td-Tomato reporter adenovirus. Here we find that not all MDA-MB-231 cancer cells have similar TGF-β mediated Smad3 transcription activity and display at least two distinct migratory populations. Importantly, Smad3 activity was significantly higher within migratory cells compared to non-migrated cells in wound healing and transwell assays. Furthermore, time-lapse experiments showed that MDA-MB-231 cells displaying Smad3 activity moved faster and a greater distance compared to cells not displaying Smad3 reporter activity. Interestingly, despite being more motile than cells with undetectable levels of Smad3 activity, high Smad3 activity was detrimental to cell motility compared to low and medium level of Smad3 activity. Conclusions: We have developed a method enabling real-time visualization of TGF-β signalling in single live cells. Breast cancer cell motility and migration is driven by sub-populations of cells with dynamic TGF-β-Smad3 activity. Those sub-populations may be responsible for tumor invasion and metastasis. [ABSTRACT FROM AUTHOR]

Tyrosine phosphorylation-dependent signaling, as mediated by members of the epidermal growth factor receptor (EGFR) family (ErbB1 to -4) of protein tyrosine kinases (PTKs), Src family PTKs (SFKs), and cytokines such as interleukin-6 (IL-6) that signal via signal transducer and activator of transcription 3 (STAT3), is critical to the development and progression of many human breast cancers. EGFR, SFKs, and STAT3 can serve as substrates for the protein tyrosine phosphatase TCPTP (PTPN2). Here we report that TCPTP protein levels are decreased in a subset of breast cancer cell lines in vitro and that TCPTP protein is absent in a large proportion of "triple-negative" primary human breast cancers. Homozygous TCPTP deficiency in murine mammary fat pads in vivo is associated with elevated SFK and STAT3 signaling, whereas TCPTP deficiency in human breast cancer cell lines enhances SFK and STAT3 signaling. On the other hand, TCPTP reconstitution in human breast cancer cell lines severely impaired cell proliferation and suppressed anchorage-independent growth in vitro and xenograft growth in vivo. These studies establish TCPTP's potential to serve as a tumor suppressor in human breast cancer. [ABSTRACT FROM AUTHOR]

The latent transcription factor Stat3 is activated by gp130, the common receptor for the interleukin (IL)-6 cytokine family and other growth factor and cytokine receptors. Ligand-induced dimerization of gp130 leads to activation of the Stat1, Stat3 and Shp2-Ras-Erk signaling pathways. Here we assess genetically the contribution of exaggerated Stat3 activation to the phenotype of gp130 Y757F/Y757F mice, in which a knock-in mutation disrupts the negative feedback mechanism on gp130-dependent Stat signaling. Compared to gp130 Y757F/Y757F mice, reduced Stat3 activation in gp130 Y757F/Y757F Stat3+/− mice increased their lifespan, prevented splenomegaly, normalized exaggerated hepatic acute-phase response and lymphocyte trafficking, and suppressed the growth of spontaneously arising gastric adenomas in young mice. These lesions share histological features of gastric polyps in aging mice with monoallelic null mutations in Smad4, which encodes the common transducer for transforming growth factor (TGF)-β signaling. Indeed, hyperactivation of Stat3 desensitizes gp130 Y757F/Y757F cells to the cytostatic effect of TGF-β through transcriptional induction of inhibitory Smad7, thereby providing a novel link for cross-talk between Stat and Smad signaling in gastric homeostasis. [ABSTRACT FROM AUTHOR]

The type III transforming growth factor β (TGFβ) receptor (TβRIII) binds both TGFβ and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of TβRIII signaling in vivo is not known. In this study, we have sought to determine the role of TβRIII during development. We identified the predominant expression sites of TβRIII mRNA as liver and heart during midgestation and have disrupted the murine TβRIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in TβRIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that TβRIII is required during murine somatic development. To assess the effects of the absence of TβRIII on the function of its ligands, primary fibroblasts were generated from TβRIII-null and wild-type embryos. Our results indicate that TβRIII deficiency differentially affects the activities of TGFβ ligands. Notably, TβRIII-null cells exhibited significantly reduced sensitivity to TGFβ2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that TβRIII is an important modulator of TGFβ2 function in embryonic fibroblasts and that reduced sensitivity to TGFβ2 may underlie aspects of the TβRIII mutant phenotype. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) plays an important role in many types of human cancers. Receptor amplification, autocrine activation and/or deletion of exons 2-7 of EGFR gene have all been associated with tumor development. The traditional model of EGFR activation via ligand induced dimerization and consequential kinase activation does not provide full understanding of its tumorigenicity. The main function of the receptor extracellular domain (ECD) has been thought to be ligand recognition and binding. We report that the EGFR ECD, through its association also negatively regulates the activity of the intracellular kinase in the absence of ligand. Even in the absence of its ligands, the EGF receptor forms homodimers, however, the ECD prevents constitutive receptor kinase activation through its intrinsic ligand-independent interaction. The removal of this domain, either partial or total, results in constitutive activation of the receptor kinase as observed by its phosphorylation in intact cells. Furthermore, EGF receptors truncated in the ECD induce phosphorylation of the wild-type full-length receptor, indicating an inter-molecular inhibitory mechanism by the receptor ECD. The tumor associated Δ2-7EGFR mutant also dimerizes with and phosphorylates the wild type EGFR in the absence of ligand. Thus, in addition to its role in ligand recognition, EGFR ECD interacts with each other, imposing an inhibitory effect on the activation of the intracellular kinase. [ABSTRACT FROM AUTHOR]

The article discusses two reports published within the issue which include one by T. A. Hirani et al. on the phosphorylation of pyruvate dehyrogenase with pyruvate dehydrogenase kinase and another one by R. S. Lee et al. on the substrate specificity of the various isoforms of Akt.

Presents a study which investigated the role of Smad signaling pathways in advanced glycation end (AGE)-mediated diabetic renal and vascular complications. Activation of Smad signaling in human diabetic nephropathy; Intracellular mechanisms involved in the activation of Smad signaling; Physiological relevance of AGE-induced ERK/p-38 MAP kinase-Smad signaling.

Two broad categories of extracellular vesicles (EVs), exosomes and shed microvesicles (sMVs), which differ in size distribution as well as protein and RNA profiles, have been described. EVs are known to play key roles in cell-cell communication, acting proximally as well as systemically. This Review discusses the nature of EV subtypes, strategies for isolating EVs from both cell-culture media and body fluids, and procedures for quantifying EVs. We also discuss proteins selectively enriched in exosomes and sMVs that have the potential for use as markers to discriminate between EV subtypes, as well as various applications of EVs in clinical diagnosis. [ABSTRACT FROM AUTHOR]

Abstract Background Bone morphogenetic proteins (BMPs) have been reported to maintain epithelial integrity and to antagonize the transforming growth factor β (TGFβ)-induced epithelial to mesenchymal transition. The expression of soluble BMP antagonists is dysregulated in cancers and interrupts proper BMP signaling in breast cancer. Methods In this study, we mined the prognostic role of BMP antagonists GREMLIN 1 (GREM1) in primary breast cancer tissues using in-house and publicly available datasets. We determined which cells express GREM1 RNA using in situ hybridization (ISH) on a breast cancer tissue microarray. The effects of Grem1 on the properties of breast cancer cells were assessed by measuring the mesenchymal/stem cell marker expression and functional cell-based assays for stemness and invasion. The role of Grem1 in breast cancer-associated fibroblast (CAF) activation was measured by analyzing the expression of fibroblast markers, phalloidin staining, and collagen contraction assays. The role of Grem1 in CAF-induced breast cancer cell intravasation and extravasation was studied by utilizing xenograft zebrafish breast cancer (co-) injection models. Results Expression analysis of clinical breast cancer datasets revealed that high expression of GREM1 in breast cancer stroma is correlated with a poor prognosis regardless of the molecular subtype. The large majority of human breast cancer cell lines did not express GREM1 in vitro, but breast CAFs did express GREM1 both in vitro and in vivo. Transforming growth factor β (TGFβ) secreted by breast cancer cells, and also inflammatory cytokines, stimulated GREM1 expression in CAFs. Grem1 abrogated bone morphogenetic protein (BMP)/SMAD signaling in breast cancer cells and promoted their mesenchymal phenotype, stemness, and invasion. Moreover, Grem1 production by CAFs strongly promoted the fibrogenic activation of CAFs and promoted breast cancer cell intravasation and extravasation in co-injection xenograft zebrafish models. Conclusions Our results demonstrated that Grem1 is a pivotal factor in the reciprocal interplay between breast cancer cells and CAFs, which promotes cancer cell invasion. Targeting Grem1 could be beneficial in the treatment of breast cancer patients with high Grem1 expression.

Sclerotium rolfsii, which causes southern blight in a wide variety of crops, is a devastating plant pathogen worldwide. Mycoviruses that induce hypovirulence in phytopathogenic fungi are potential biological control resources against fungal plant diseases. However, in S. rolfsii, mycoviruses are rarely reported. In a previous study, we found a hypovirulent strain carrying a diverse pattern of dsRNAs. Here, we utilized the RNA_Seq technique to detect viral sequences. Deep sequencing, RT-PCR and Sanger sequencing validation analyses revealed that this strain harbors various new viral species that show affinity to the distinctly established and proposed families Benyviridae, Endornaviridae, Fusariviridae, Hypoviridae, and Fusagraviridae. Moreover, some viral sequences that could not be assigned to any of the existing families or orders were also identified and showed similarities to the Alphavirus, Ourmiavirus, phlegivirus-like and Curvularia thermal tolerance virus-like groups. In addition, we also conducted deep sequencing analysis of small RNAs in the virus-infecting fugal strain. The results indicated that the Dicer-mediated gene silencing mechanism was present in S. rolfsii. This is the first report of viral diversity in a single S. rolfsii fungal strain, and the results presented herein might provide insight into the taxonomy and evolution of mycoviruses and be useful for the exploration of mycoviruses as biocontrol agents.

Here, the molecular characterization of a novel mycovirus that was isolated from a phytopathogenic fungus Magnaporthe oryzae and designed as Magnaporthe oryzae ourmia-like virus 4 (MOLV4) is reported. MOLV4 has a genome that is 2497 bp long and possesses a single open reading frame (ORF), which encodes the product RNA-dependent RNA polymerase (RdRp). Sequence similarities were found between the MOLV4 encoded RdRp and the counterparts of a few previously reported ourmia-like mycoviruses. Virus-curing and biological comparison indicate that the virus has no or mild effects on the morphology and mycelium growth rate of the host fungus. Phylogenetic analysis using the RdRp aa sequences was performed. The results show that MOLV4 is clustered with the ourmia-like mycoviruses, forming a clade closely related to ourmiaviruses but distinct from narnaviruses. In addition, database searches revealed that several MOLV4-related sequences are present in the transcriptome shotgun assembly (TSA) library, expressed sequence tag database (ESTdb), whole-genome shotgun (WGS) library, and genomic survey sequences (GSS) libraries of a few other species of eukaryote organisms. Our results show that MOLV4, together with other similar ourmia-like mycoviruses, might represent a virus clade that links the plant ourmiaviruses and fungal narnaviruses and has a wide range of hosts.

Three dsRNAs, in sizes of approximately 2.5–5 kbp, were detected in the plant pathogenic fungus Nigrospora oryzae strain CS-7.5-4. Genomic analysis showed that the 5.0 kb dsRNA was a victorivirus named as Nigrospora oryzae victorivirus 2 (NoRV2). The genome of NoRV2 was 5166 bp in length containing two overlapping open reading frames (ORFs), ORF1 and ORF2. ORF1 was deduced to encode a coat protein (CP) showing homology to the CPs of viruses belonging to the Totiviridae family. The stop codon of ORF1 and the start codon of ORF2 were overlapped by the tetranucleotide sequence AUGA. ORF2 was predicted to encode an RNA-dependent RNA polymerase (RdRp), which was highly similar to the RdRps of victoriviruses. Virus-like particle examination demonstrated that the genome of NoRV2 was solely encapsidated by viral particles with a diameter of approximately 35 nm. The other two dsRNAs that were less than 3.0 kb were predicted to be the genomes of two mitoviruses, named as Nigrospora oryzae mitovirus 1 (NoMV1) and Nigrospora oryzae mitovirus 2 (NoMV2). Both NoMV1 and NoMV2 were A-U rich and with lengths of 2865 and 2507 bp, respectively. Mitochondrial codon usage inferred that each of the two mitoviruses contains a major large ORF encoding a mitoviral RdRp. Horizontal transfer experiments showed that the NoMV1 and NoMV2 could be cotransmitted horizontally via hyphal contact to other virus-free N. oryzae strains and causes phenotypic change to the recipient, such as an increase in growth rate. This is the first report of mitoviruses in N. oryzae.

Mycoviruses associated with hypovirulence are potential biological control agents and could be useful to study the pathogenesis of fungal host pathogens. Sclerotium rolfsii, a pathogenic fungus, causes southern blight in a wide variety of crops. In this study, we isolated a series of dsRNAs from a debilitated S. rolfsii strain, BLH-1, which had pronounced phenotypic aberrations including reduced pathogenicity, mycelial growth and deficient sclerotia production. Virus-curing and horizontal transmission experiments that eliminated or transmitted, respectively, all dsRNA elements showed that the dsRNAs were involved in the hypovirulent traits of BLH-1. Ultrastructure examination also showed hyphae fracture and cytoplasm or organelle degeneration in BLH-1 hyphal cells compared to the virus-free strain. Three assembled cDNA contigs generated from the cDNA library cloned from the purified dsRNA indicated that strain BLH-1 was infected by at least three novel mycoviruses. One has similarity to the hypovirulence-associated Sclerotinia sclerotiorum hypovirus 2 (SsHV2) in the family Hypoviridae, and the other two are related to two different unclassified dsRNA mycovirus families. To our knowledge, this is the first report of S. rolfsii hypovirulence that was correlated with its associated dsRNA.

Appropriate cellular signaling is essential to control cell proliferation, differentiation, and cell death. Aberrant signaling can have devastating consequences and lead to disease states, including cancer. The transforming growth factor-β (TGF-β) signaling pathway is a prominent signaling pathway that has been tightly regulated in normal cells, whereas its deregulation strongly correlates with the progression of human cancers. The regulation of the TGF-β signaling pathway involves a variety of physiological regulators. Many of these molecules act to alter the activity of Smad proteins. In contrast, the number of molecules known to affect the TGF-β signaling pathway at the receptor level is relatively low, and there are no known direct modulators for the TGF-β type II receptor (TβRII). Here we identify SPSB1 (a Spry domain-containing Socs box protein) as a novel regulator of the TGF-β signaling pathway. SPSB1 negatively regulates the TGF-β signaling pathway through its interaction with both endogenous and overexpressed TβRII (and not TβRI) via its Spry domain. As such, TβRII and SPSB1 co-localize on the cell membrane. SPSB1 maintains TβRII at a low level by enhancing the ubiquitination levels and degradation rates of TβRII through its Socs box. More importantly, silencing SPSB1 by siRNA results in enhanced TGF-β signaling and migration and invasion of tumor cells. [ABSTRACT FROM AUTHOR]

Spherical virus-like particles about 40 nm in diameter were observed under transmission electron microscope (TEM) and two dsRNA bands (dsRNA-1 and dsRNA-2) were detected on agarose gel after extraction from the mycelial preparation of a Colletotrichum acutatum strain HNZJ001 that isolated from an anthracnose lesion on immature pepper fruit. The complete nucleotide sequences of the dsRNAs were determined. DsRNA-1 (1762 nt) and dsRNA-2 (1381 nt) each contained a single open reading frame and potentially encoded 62 kDa and 40 kDa proteins, respectively. The 62 kDa protein showed similarity to the RNA-dependent RNA polymerase (RdRp) of partitiviruses, while the 40 kDa product had no significant similarity to any published capsid protein throughout all databases, besides of low homology with the hypothetical "capsid" protein of a few partitiviruses in fungus Ustilaginoidea virens. Genome comparison and phylogenetic analysis indicated that the virus is closely related to the mycovirus in the family Partitiviridae. The results suggested a novel two-segment dsRNA virus be detected. We name it Colletotrichum acutatum partitivirus 1 (CaPV1). RT-PCR detection, using a primer pair based on the RdRp of the dsRNA-1 showed very high efficiency of CaPV1 transmission into the progenies of the fungus. Virus curing and fungal phenotype observation for evaluation of the impact of CaPV1 in host fungus were also carried out. [ABSTRACT FROM AUTHOR]

Activation of the canonical TGF-β signaling pathway provides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but to what extent mutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2, and SMAD3 and analyzed for allelic loss by single-nucleotide polymorphism (SNP) microarray analysis. Mutation spectra were compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3 mutations in sporadic CRCs was 8.6% (64 of 744), 3.4% (25 of 744), and 4.3% (32 of 744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4 mutations were associated with mucinous histology. The mutation spectra of SMAD2 and SMAD3 were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation-regulated Ser-Ser-X- Ser motifs within SMAD2 and SMAD3. Functional analyses of selected mutations showed reductions in SMAD3 transcriptional activity and SMAD2-SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented and mutually exclusive to SMAD4 mutation, underlining the critical roles of these three proteins within the TGF-β signaling pathway. [ABSTRACT FROM AUTHOR]

Recent crystallographic data on the isolated extracellular domain of the epidermal growth factor receptor (EGFR) have suggested a model for its activation by ligand. We have tested this model in the context of the full-length EGFR displayed at the cell surface, by introducing mutations in two regions (CR1 and CR2) of the extracellular domain thought to be critical for regulation of receptor activation. Mutations in the CR1 and CR2 domains have opposing effects on ligand binding affinity, receptor dimerization, tyrosine kinase activation, and signaling competence. Tyr246 is a critical residue in the CR1 loop, which is implicated in the positioning and stabilization of the receptor dimer interface after ligand binding; mutations of Tyr246 impair or abolish receptor function. Mutations in CR2, which weaken the interaction that restricts the receptor to the tethered (inactive) state, enhance responsiveness to EGF by increasing affinity for the ligand. However, weakening of the CR1/CR2 interaction does not result in spontaneous activation of the receptors' kinase. We have used an antibody (mAb 806), which recognizes a transition state of the EGF receptor between the negatively constrained, tethered state and the fully active back-to-back dimer conformation, to follow conformational changes in the wild-type and mutant EGF receptors after ligand binding. Our results suggest that EGFR on the cell surface can be untethered, but this form is inactive; thus, untethering of the receptor is not sufficient for activation, and ligand binding is essential for the correct positioning of the two receptor subunits to achieve kinase activation. [ABSTRACT FROM AUTHOR]

Examines the crystal structure of a truncated epidermal growth factor receptor (EGFR) extracellular domain. Biochemistry of the EGFR system; Role of ligand-induced receptor dimers in the EGFR signaling system; Mechanism of receptor dimerization.