Activation Of Signal Transduction Pathways Research Articles

Insights into functional connectivity in mammalian signal transduction pathways by pairwise comparison of protein interaction partners of critical signaling hubs.

Growth factors and cytokines activate signal transduction pathways and regulate gene expression in eukaryotes. Intracellular domains of activated receptors recruit several protein kinases as well as transcription factors that serve as platforms or hubs for the assembly of multi-protein complexes. The signaling hubs involved in a related biologic function often share common interaction proteins and target genes. This functional connectivity suggests that a pairwise comparison of protein interaction partners of signaling hubs and network analysis of common partners and their expression analysis might lead to the identification of critical nodes in cellular signaling. A pairwise comparison of signaling hubs across several related pathways might reveal novel signaling modules. Analysis of protein interaction connectome by Venn (PIC-Venn) of transcription factors STAT1, STAT3, NFKB1, RELA, FOS, and JUN, and their common interaction network suggested that BRCA1 and TSC22D3 function as critical nodes in immune responses by connecting the signaling hubs into signaling modules. Transcriptional regulation of critical hubs may play a major role in the lung epithelial cells in response to SARS-CoV-2 and in COVID-19 patients. Mutations and differential expression levels of these critical nodes and modules in pathological conditions might deregulate signaling pathways and their target genes involved in inflammation. Biological connectivity emerges from the structural connectivity of interaction networks across several signaling hubs in related pathways. The main objectives of this study are to identify critical hubs, critical nodes, and modules involved in the signal transduction pathways of innate and adaptive immunity. Application of PIC-Venn to several signaling hubs might reveal novel nodes and modules that can be targeted by small regulatory molecules to simultaneously activate or inhibit cell signaling in health and disease.

Cytokine chemokine network in tumor microenvironment: Impact on CSC properties and therapeutic applications

A subpopulation of cells in many cancers has stem cell traits, mediates metastasis, and contributes to treatment resistance. These cells are considered as cancer stem cells (CSCs). CSC properties of tumor cells are immensely regulated by close interactions with tumor microenvironment components such as mesenchymal stem cells, tumor related fibroblasts, adipocytes, endothelial cells, and immune cells via the intricate network of cytokines, chemokines, and growth factors. Inflammatory cytokines including interleukin (IL)-1, IL-6, and IL-8 play a major role in these interactions via the activation of signal transduction pathways like Stat3/NF-κB etc. in stromal and tumor cells. The activation of these pathways increases the release of more cytokines, resulting in positive feedback loops which help in CSC self-renewal. The pathways controlled by these cytokine loops are similar to those that are active during chronic inflammation and wound healing, suggesting that they might have critical role in establishing relationship between inflammation and cancer. Anti-inflammatory drugs have been identified to inhibit these cytokines and their receptor mediated pathways. These agents have the potential to target CSCs by inhibiting signals from the tumor microenvironment and considered to be a potential candidate for future therapeutics. The significance of cytokines released from the tumor microenvironment in different phases of cancer, as well as their potential application in cancer therapeutics is discussed in this article.

Class I PI3K regulatory subunits control differentiation of dendritic cell subsets and regulate Flt3L mediated signal transduction

Dendritic cells (DCs) play pivotal roles in initiating and shaping both innate and adaptive immune responses. The spatiotemporal expression of transcription factor networks and activation of specific signal transduction pathways determine the specification, distribution and differentiation of DC subsets. Even though pioneering studies have established indispensable roles for specific catalytic subunits (p110δ and p110γ) in immune cells, functions of the regulatory subunits, particularly of Class I PI3K, within the hematopoietic system remain incompletely understood. In the study presented here, we deleted the key regulatory subunits—p85α and p85β of the Class IA PI3K in hematopoietic cells and studied its impact on DC differentiation. Our studies identify that a deficiency of p85 causes increased differentiation of conventional DC (cDC) 2 and plasmacytoid DC (pDC) subsets in the spleen. On the other hand, DC numbers in the bone marrow (BM), thymus and lymph nodes were decreased in p85 mutant mice. Analysis of DC-specific progenitors and precursors indicated increased numbers in the BM and spleen of p85 deficient mice. In-vitro differentiation studies demonstrated augmented DC-differentiation capacities of p85 deficient BM cells in the presence of GM-CSF and Flt3L. BM chimera studies established that p85 deficiency affects DC development through cell intrinsic mechanisms. Molecular studies revealed increased proliferation of DCs and common DC progenitors (CDPs) in the absence of p85 and altered signal transduction pathways in p85 mutant DC subsets in response to Flt3L. In essence, data presented here, for the first time, unequivocally establish that the P85α subunit of class IA PI3Ks has an indispensable role in the development and maintenance of DCs.

6-(Methylsulfinyl) Hexyl Isothiocyanate Inhibits IL-6 and CXCL10 Production in TNF-α-Stimulated Human Oral Epithelial Cells.

6-(Methylsulfinyl) hexyl isothiocyanate (6-MSITC) is a bioactive substance found in wasabi (Wasabia japonica) and has been reported to have some bioactive effects including anticancer and antioxidant effects. However, there are no reports on its effects on periodontal resident cells, and many points remain unclear. In this study, we aimed to investigate whether 6-MSITC exerts anti-inflammatory effects on human oral epithelial cells, including effects on signal transduction pathway activation. 6-MSITC inhibited interleukin (IL)-6 and C-X-C motif chemokine ligand 10 (CXCL10) production in TNF-α-stimulated TR146 cells, which are a human oral epithelial cell line. Moreover, we found that 6-MSITC could suppress signal transducer and activator of transcription (STAT)3, nuclear factor (NF)-κB, and p70S6 kinase (p70S6K)-S6 ribosomal protein (S6) pathways activation in TNF-α-stimulated TR146 cells. Furthermore, STAT3 and NF-κB inhibitors could suppress IL-6 and CXCL10 production in TNF-α-treated TR146 cells. In summary, 6-MSITC could decrease IL-6 and CXCL10 production in human oral epithelial cell by inhibiting STAT3 and NF-κB activation.

Targeting the Holy Triangle of Quorum Sensing, Biofilm Formation, and Antibiotic Resistance in Pathogenic Bacteria

Chronic and recurrent bacterial infections are frequently associated with the formation of biofilms on biotic or abiotic materials that are composed of mono- or multi-species cultures of bacteria/fungi embedded in an extracellular matrix produced by the microorganisms. Biofilm formation is, among others, regulated by quorum sensing (QS) which is an interbacterial communication system usually composed of two-component systems (TCSs) of secreted autoinducer compounds that activate signal transduction pathways through interaction with their respective receptors. Embedded in the biofilms, the bacteria are protected from environmental stress stimuli, and they often show reduced responses to antibiotics, making it difficult to eradicate the bacterial infection. Besides reduced penetration of antibiotics through the intricate structure of the biofilms, the sessile biofilm-embedded bacteria show reduced metabolic activity making them intrinsically less sensitive to antibiotics. Moreover, they frequently express elevated levels of efflux pumps that extrude antibiotics, thereby reducing their intracellular levels. Some efflux pumps are involved in the secretion of QS compounds and biofilm-related materials, besides being important for removing toxic substances from the bacteria. Some efflux pump inhibitors (EPIs) have been shown to both prevent biofilm formation and sensitize the bacteria to antibiotics, suggesting a relationship between these processes. Additionally, QS inhibitors or quenchers may affect antibiotic susceptibility. Thus, targeting elements that regulate QS and biofilm formation might be a promising approach to combat antibiotic-resistant biofilm-related bacterial infections.

Abstract 4002: Identification of signal transduction pathway activity with potential clinical target in high-grade serous ovarian carcinoma

Abstract Background: High-grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer. It has a high mortality rate, even after successful first-line treatment with debulking surgery and chemotherapy. Although therapeutic options for targeted therapy are rapidly expanding, identification of patients who respond to these therapies remains a challenge. In recognition of the importance of the functional phenotype of cancer cells, Verhaegh et al. (Cancer Res 2014) developed assays to measure functional activity of signal transduction pathways (STPs) based on mRNA expression levels of pathway-specific target genes. In this study, we aimed to identify HGSCs with STP activity with a potential clinical target by comparing their activity with STP activity in normal Fallopian tube epithelium (FTE), the tissue of origin of most HGSCs. Methods: We included 50 primary tumor samples taken prior to start of chemotherapy of postmenopausal patients diagnosed with advanced stage HGSC and 9 morphologically normal FTE samples of healthy postmenopausal women. Using pathway assays, we assessed functional pathway activity of the androgen receptor (AR), estrogen receptor (ER), PI3K, MAPK, Hedgehog, TGFβ and Notch pathways. Differences in STP activity between groups were compared with Mann-Whitney U tests. Cut-off value for aberrant STP activity was defined as two standard deviations above the mean value of STP activity measured in FTE samples. Results: In the HGSC group we observed lower median ER (p < 0.001) pathway activity and higher median PI3K (p < 0.001), Hedgehog (p < 0.001) and TGFβ pathway (p = 0.020) activity as compared to the FTE group. In individual HGSC samples, aberrant activity was identified for the MAPK (n = 10), PI3K (n = 22), Hedgehog (n = 28) and TGFβ (n = 21) pathways. Frequently observed combinations of aberrant STP activity were Hedgehog/TGFβ (n = 12) and Hedgehog/PI3K (n = 9). In total, we identified at least one STP with potential clinical target in 88% (44/50) of HGSC samples. Conclusions: Our analysis enabled the identification of STP activity with a potential clinical target in 88% of the analyzed HGSC samples. Differentiation between normal and aberrant STP activity could have clinical utility in the selection of HGSC patients for targeted therapy. A prospective study (STAPOVER) has been designed to demonstrate clinical utility. Citation Format: Phyllis Van der Ploeg, Yvonne J.W. Wesseling-Rozendaal, Eveline C. Biezen-Timmermans, Jurgen M.J. Piek. Identification of signal transduction pathway activity with potential clinical target in high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4002.

Abstract 403: Analysis of RET solvent front mutations shows an effect on protein activity in tumor cells

Abstract The rearranged during transfection (RET) gene encodes a receptor tyrosine kinase that is involved in the activation of several signal transduction pathways. Various RET fusions and point mutations were shown to play a role as oncogene drivers in different cancers. In 2020, two selective RET tyrosine kinase inhibitors (TKIs), pralsetinib and selpercatinib, were approved by the FDA for treatment of RET fusion-positive non-small cell lung cancer, RET-mutant medullary thyroid cancer and RET fusion-positive thyroid cancer. These drugs demonstrate a significant advance in efficacy and safety over previous, multi-targeted TKIs for the treatment of RET-positive patients. However, development of acquired resistance to these agents has been observed through the appearance of on-target secondary solvent front mutations in the RET G810 residue. Moreover, 5 different types of amino acid changes - G810A/C/R/S/V have been identified in this position, of which G810C, G810R and G810S are better characterized but their effect on protein activity is lacking. We used a high-throughput functional assay, in which the mutated protein is expressed together with a fluorescently labeled reporter in a cell-based assay. Using this system, we systematically assessed the activity of different combinations of RET fusions or oncogenic point mutations with one of three different common solvent front mutations and compared them to the activity of the individual mutations. Our results show that when a solvent front mutation is expressed on a RET oncogenic point mutation background, the solvent front mutation consistently decreases the oncogenic protein activity. This is supported by our observation that the individual solvent front mutations expressed on a RET WT background show an activity that is lower than that of the RET WT protein. On a RET fusion background the effect of solvent front mutations is more variable, with some RET fusion-solvent front mutation combinations showing a decreased activity and some not affecting the activity of the fusion protein. Across all mutations and fusions tested, the G810R and G810C mutations had a larger effect on RET activity than that of the G810S mutation. We also compared the effect of drug treatment (pralsetinib and selpercatinib) on RET oncogenic activity in the presence of the different solvent front mutations and observed that on all mutational backgrounds tested, the solvent front mutations have an effect on the response dynamics to the drug, and that the extent of the effect depends on the type of solvent front mutation, with G810R and G810C decreasing sensitivity to drug treatment to a larger extent compared to G810S. Together, our findings provide a better understanding of the differential effects of solvent front mutations on RET activity under different oncogenic contexts. Citation Format: Shay Rotkopf, DIkla Haham, Lea Birnbaum, Zohar Barbash, Gabi Tarcic. Analysis of RET solvent front mutations shows an effect on protein activity in tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 403.

Abstract 2437: Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53

Abstract Human breast cancers often overexpress MDM family proteins MDM2 and MDMX(MDM4). MDM2 is an E3 ubiquitin ligase and MDMX is an E4 that promotes ubiquitination of MDM2 targets (however this is an understudied area). The Cancer Genome Atlas (TCGA) shows high MDMX/MDM2 expression in all breast cancer subtypes. We identified that MDMX depletion reduces circulating tumor cells (CTCs) and metastasis of breast cancers with mtp53. Our ongoing objective is to determine the mechanisms of how MDMX promotes CTCs and metastasis, and the potential MDMX regulated ubiquitinated targets. In TNBC primary tumors, knockdown of mdmx correlates with reduced expression of the G protein coupled receptor, CXCR4. We used mtp53 expressing T47D ER+, and MDA-MB-468, and MDA-MB-231 metastatic TNBC cells and their derived CTC lines, and shRNA-mediated knockdown of MDM2 and MDMX derived lines, to address the potential MDMX targets. We assessed metastatic properties of migration and invasion changed by the knockdown of MDM2/MDMX in parental and CTC cell lines using multi-faceted cell biological and biochemical approaches. Depletion of MDM2 in T47D and MDA-MB-231 cells increased the level of MDMX, demonstrating a critical function of MDM2 for regulating MDMX. MDMX depletion surprisingly did not change MDM2 levels, but did reduce the migration of both MDA-MB-231 cells and their derived CTC lines. Interestingly, the parental MDA-MB-231 cells migrated more rapidly in scratch assays, compared to both their derived CTCs and MDMX-depleted CTCs, suggesting migration to be critical for intravasation, but not for latter cancer cell survival. Experiments are in progress to address if loss of MDMX perturbs CXCL12 ligand communication with the CXCR4 receptor in parental and CTC breast cancer lines. Preliminary data suggests activated MDMX expression correlates with the activation of these metastasis signal transduction pathways. The ubiquitination targets of MDMX/MDM2 in the parental and CTC cell lines are being defined by affinity purification in the presence, and absence, of MDMX/MDM2, and add back of purified MDMX/MDM2 to validate targets. As expected, purified MDMX increases MDM2 E3 ubiquitin ligase activity, and on its own MDMX is inert. Stable isotope labeling in cell culture suggested protein targets involved in DNA replication and repair pathways. This preliminary data informed our interest in studying DNA damage signaling by pharmacologically inhibiting the DNA repair protein PARP. We examined how MDMX/MDM2 pathways respond to DNA damage caused by PARP inhibition. Our preliminary data affirms regulation of the MDMX/MDM2 stress-response circuit by replication stress response pathways, and reveals the requirement of MDMX/MDM2 for their function in the studied cell lines. Disrupting MDMX function has potential to be beneficial for inhibition of breast cancer progression and metastasis. Supported by BCRF-20-011. Citation Format: Rusia Lee, Viola Ellison, Gu Xiao, Dominique Forbes, Pam Leybengrub, Alexandra Kern, Falande Alexandre, George Annor, Jill Bargonetti. Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2437.

Towards Understanding the Relationship Between ER Stress and Unfolded Protein Response in Amyotrophic Lateral Sclerosis.

Biological stress due to the aberrant buildup of misfolded/unfolded proteins in the endoplasmic reticulum (ER) is considered a key reason behind many human neurodegenerative diseases. Cells adapted to ER stress through the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by degeneration of the motor system. It has largely been known that ER stress plays an important role in the pathogenesis of ALS through the dysregulation of proteostasis. Moreover, accumulating evidence indicates that ER stress and UPR are important players in TDP-43 pathology. In this mini-review, the complex interplay between ER stress and the UPR in ALS and TDP-43 pathology will be explored by taking into account the studies from in vitro and in vivo models of ALS. We also discuss therapeutic strategies to control levels of ER stress and UPR signaling components that have contrasting effects on ALS pathogenesis.

The Rationale for "Laser-Induced Thermal Therapy (LITT) and Intratumoral Cisplatin" Approach for Cancer Treatment.

Cisplatin is one of the most widely used anticancer drugs in the treatment of various types of solid human cancers, as well as germ cell tumors, sarcomas, and lymphomas. Strong evidence from research has demonstrated higher efficacy of a combination of cisplatin and derivatives, together with hyperthermia and light, in overcoming drug resistance and improving tumoricidal efficacy. It is well known that the antioncogenic potential of CDDP is markedly enhanced by hyperthermia compared to drug treatment alone. However, more recently, accelerators of high energy particles, such as synchrotrons, have been used to produce powerful and monochromatizable radiation to induce an Auger electron cascade in cis-platinum molecules. This is the concept that makes photoactivation of cis-platinum theoretically possible. Both heat and light increase cisplatin anticancer activity via multiple mechanisms, generating DNA lesions by interacting with purine bases in DNA followed by activation of several signal transduction pathways which finally lead to apoptosis. For the past twenty-seven years, our group has developed infrared photo-thermal activation of cisplatin for cancer treatment from bench to bedside. The future development of photoactivatable prodrugs of platinum-based agents injected intratumorally will increase selectivity, lower toxicity and increase efficacy of this important class of antitumor drugs, particularly when treating tumors accessible to laser-based fiber-optic devices, as in head and neck cancer. In this article, the mechanistic rationale of combined intratumor injections of cisplatin and laser-induced thermal therapy (CDDP–LITT) and the clinical application of such minimally invasive treatment for cancer are reviewed.

New Targets for Antiviral Therapy: Inhibitory Receptors and Immune Checkpoints on Myeloid Cells.

Immune homeostasis is achieved by balancing the activating and inhibitory signal transduction pathways mediated via cell surface receptors. Activation allows the host to mount an immune response to endogenous and exogenous antigens; suppressive modulation via inhibitory signaling protects the host from excessive inflammatory damage. The checkpoint regulation of myeloid cells during immune homeostasis raised their profile as important cellular targets for treating allergy, cancer and infectious disease. This review focuses on the structure and signaling of inhibitory receptors on myeloid cells, with particular attention placed on how the interplay between viruses and these receptors regulates antiviral immunity. The status of targeting inhibitory receptors on myeloid cells as a new therapeutic approach for antiviral treatment will be analyzed.

GPR83 Engages Endogenous Peptides from Two Distinct Precursors to Elicit Differential Signaling

PEN is an abundant neuropeptide that activates GPR83, a G protein-coupled receptor that is considered a novel therapeutic target due to its roles in regulation of feeding, reward, and anxiety-related behaviors. The major form of PEN in the brain is 22 residues in length. Previous studies have identified shorter forms of PEN in mouse brain and neuroendocrine cells; these shorter forms were named PEN18, PEN19 and PEN20, with the number reflecting the length of the peptide. The C-terminal five residues of PEN20 are identical to the C-terminus of a procholecystokinin (proCCK)-derived peptide, named proCCK56-62, that is present in mouse brain. ProCCK56-62 is highly conserved across species although it has no homology to the bioactive cholecystokinin domain. ProCCK56-62 and a longer form, proCCK56-63 were tested for their ability to engage GPR83. Both peptides bind GPR83 with high affinity, activate second messenger pathways, and induce ligand-mediated receptor endocytosis. Interestingly, the shorter PEN peptides, ProCC56-62, and ProCCK56-63 differentially activate signal transduction pathways. Whereas PEN22 and PEN20 facilitate receptor coupling to Gai, PEN18, PEN19 and ProCCK peptides facilitate coupling to Gas. Furthermore, the ProCCK peptides exhibit dose dependent Ga subtype selectivity in that they faciliate coupling to Gas at low concentrations and Gai at high concentrations. These data demonstrate that peptides derived from two distinct peptide precursors can differentially activate GPR83, and that GPR83 exhibits Ga subtype preference depending on the nature and concentration of the peptide. These results are consistent with the emerging idea that endogenous neuropeptides function as biased ligands. Significance Statement We found that peptides derived from proCCK bind and activate GPR83, a G protein-coupled receptor that is known to bind peptides derived from proSAAS. Different forms of the proCCK- and proSAAS-derived peptides show biased agonism, activating Gas or Gai depending on the length of the peptide and/or its concentration.

Modification of Ischemia/Reperfusion-Induced Alterations in Subcellular Organelles by Ischemic Preconditioning.

It is now well established that ischemia/reperfusion (I/R) injury is associated with the compromised recovery of cardiac contractile function. Such an adverse effect of I/R injury in the heart is attributed to the development of oxidative stress and intracellular Ca2+-overload, which are known to induce remodeling of subcellular organelles such as sarcolemma, sarcoplasmic reticulum, mitochondria and myofibrils. However, repeated episodes of brief periods of ischemia followed by reperfusion or ischemic preconditioning (IP) have been shown to improve cardiac function and exert cardioprotective actions against the adverse effects of prolonged I/R injury. This protective action of IP in attenuating myocardial damage and subcellular remodeling is likely to be due to marked reductions in the occurrence of oxidative stress and intracellular Ca2+-overload in cardiomyocytes. In addition, the beneficial actions of IP have been attributed to the depression of proteolytic activities and inflammatory levels of cytokines as well as the activation of the nuclear factor erythroid factor 2-mediated signal transduction pathway. Accordingly, this review is intended to describe some of the changes in subcellular organelles, which are induced in cardiomyocytes by I/R for the occurrence of oxidative stress and intracellular Ca2+-overload and highlight some of the mechanisms for explaining the cardioprotective effects of IP.

Measurement of activity of developmental signal transduction pathways to quantify stem cell pluripotency and phenotypically characterize differentiated cells

Important challenges in stem cell research and regenerative medicine are reliable assessment of pluripotency state and purity of differentiated cell populations. Pluripotency and differentiation are regulated and determined by activity of developmental signal transduction pathways (STPs). To date activity of these STPs could not be directly measured on a cell sample.Here we validate a novel assay platform for measurement of activity of developmental STPs (STP) for use in stem cells and stem cell derivatives. In addition to previously developed STP assays, we report development of an additional STP assay for the MAPK-AP1 pathway. Subsequently, activity of Notch, Hedgehog, TGFβ, Wnt, PI3K, MAPK-AP1, and NFκB signaling pathways was calculated from Affymetrix transcriptome data of human pluripotent embryonic (hES) and iPS cell lines under different culture conditions, organ-derived multipotent stem cells, and differentiated cell types, to generate quantitative STP activity profiles.Results show that the STP assay technology enables reliable and quantitative measurement of multiple STP activities simultaneously on any individual cell sample. Using the technology, we found that culture conditions dominantly influence the pluripotent stem cell STP activity profile, while the origin of the stem cell line was a minor variable. A pluripotency STP activity profile (Pluripotency qPAP) was defined (active PI3K, MAPK, Hedgehog, Notch, TGFβ, and NFκB pathway, inactive Wnt pathway). Differentiation of hES cells to intestinal progenitor cells resulted in an STP activity profile characterized by active PI3K, Wnt and Notch pathways, comparable to the STP activity profile measured on primary intestinal crypt stem cells. Quantitative STP activity measurement is expected to improve experimental reproducibility and standardization of pluripotent and multipotent stem cell culture/differentiation, and enable controlled manipulation of pluripotency/differentiation state using pathway targeting compounds.

Blocking the Notch signal transduction pathway promotes tumor growth in breast cancer by promoting the expression of suppressible inflammatory factors.

BackgroundBreast cancer is the most common malignant tumor among all female tumors. It seriously affects the health and lives of patients, and poses a significant economic burden. The study of the molecular mechanisms of breast cancer occurrence, proliferation and growth and development is of great clinical significance.MethodsNotch1 knockout mice were obtained by gene targeting. The expression of inflammatory factor arginase-1 in each group of tumors was observed by immunofluorescence staining. Semi-quantitative detection of Notch1, Arginase-1, and proteins belonging to the PI3K-AKT pathway by western blot. The expression level of interleukin-3 (IL-3), and IL-4 in serum was quantified by enzyme linked immunosorbent assay (ELISA).ResultsIn this study, Notch1 knockout in mice promoted the cell proliferation of breast cancer. Further study on molecular mechanisms demonstrated that the increased cell proliferation resulted from the activation of the PI3K-AKT signal transduction pathway. In addition, the expression of the M2-type inflammatory factor arginase-1 significantly increased, which was dependent on the activation of the PI3K-AKT pathway, indicating that Notch1 knockout in mice promoted the polarization of tumor-associated macrophages (TAMs). Consistent with this, IL-3 and IL-4 expression also significantly increased in the serum of Notch1 knockout mice.ConclusionsAccording to our results, Notch1 knockout in mice significantly promoted the cell proliferation of breast cancer, not only by activating the PI3K-AKT pathway, but also by promoting the polarization of TAMs towards the M2-type phenotype.

STIM2 promotes the invasion and metastasis of breast cancer cells through the NFAT1/TGF-β1 pathway.

A large amount of evidence indicates that the abnormal activation of multiple signal transduction pathways in cells is closely related to the occurrence and development of tumors. TGF-β and NFAT1 signaling pathways can inhibit cell proliferation and promote apoptosis in the early stage of breast cancer, but with the increase of tumor malignancy, the two appear to promote tumor progression and deterioration. Therefore, the study of the relationship between STIM2 and NFAT1/TGF-β1 is helpful for the discovery and treatment of breast cancer, which is of great significance for improving the survival rate of breast cancer patients. This article focuses on the effect of STIM2 molecules on breast cancer cell migration through the NFAT1/ TGF-β1 pathway and discusses the regulatory mechanism of STIM2 affecting breast cancer cell migration. Experimental data shows that the positive rate of breast cancer NFAT1 is 54%, which is significantly lower than that of benign breast Tissue 85%; the positive expression rate of TGF-β1 in benign breast tissue is 85%, and the positive expression rate in breast cancer tissue is 49%. The results show that STIM2 protein can promote the invasion and metastasis of breast cancer cells through the NFAT1 / TGF-β1 pathway.

Organ defects of the Usp7K444R mutant mouse strain indicate the essential role of K63-polyubiquitinated Usp7 in organ formation

BackgroundK63-linked polyubiquitination of proteins have nonproteolytic functions and regulate the activity of many signal transduction pathways. USP7, a HIF1α deubiquitinase, undergoes K63-linked polyubiquitination under hypoxia. K63-polyubiquitinated USP7 serves as a scaffold to anchor HIF1α, CREBBP, the mediator complex, and the super elongation complex to enhance HIF1α-induced gene transcription. However, the physiological role of K63-polyubiquitinated USP7 remains unknown. MethodsUsing a Usp7K444R point mutation knock-in mouse strain, we performed immunohistochemistry and standard molecular biological methods to examine the organ defects of liver and kidney in this knock-in mouse strain. Mechanistic studies were performed by using deubiquitination, immunoprecipitation, and quantitative immunoprecipitations (qChIP) assays. ResultsWe observed multiple organ defects, including decreased liver and muscle weight, decreased tibia/fibula length, liver glycogen storage defect, and polycystic kidneys. The underlying mechanisms include the regulation of protein stability and/or modulation of transcriptional activation of several key factors, leading to decreased protein levels of Prr5l, Hnf4α, Cebpα, and Hnf1β. Repression of these crucial factors leads to the organ defects described above. ConclusionsK63-polyubiquitinated Usp7 plays an essential role in the development of multiple organs and illustrates the importance of the process of K63-linked polyubiquitination in regulating critical protein functions.

Signal transduction pathway activity in high-grade serous carcinoma, its precursors and Fallopian tube epithelium

ObjectiveTo determine the activity of key signal transduction pathways in serous tubal intraepithelial carcinoma (STIC) and concurrent high-grade serous carcinoma (HGSC) and compare this to pathway activity in normal Fallopian tube epithelium (FTE). MethodsWe assessed mRNA expression levels of pathway-specific target genes with RT-qPCR in STIC and concurrent HGSC (n = 8) and normal FTE (n = 8). Subsequently, signal transduction pathway assays were used to assess functional activity of the androgen (AR) and estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor beta (TGF-β) and canonical wingless-type MMTV integration site (Wnt) pathways. ResultsThere were no statistically significant differences in pathway activity between STIC and HGSC, but STIC and HGSC demonstrated significantly lower ER and higher PI3K and HH pathway activity in comparison to normal FTE, suggesting these pathways as putative early drivers. In addition, we determined FOXO3a protein expression by immunohistochemistry and found loss of FOXO3a protein expression in STIC and HGSC compared to normal FTE. This observation confirmed that activation of PI3K signaling by loss of FOXO is an early hallmark of serous carcinogenesis. Furthermore, HGSC demonstrated significant loss of AR and Wnt pathway activity in relation to FTE, suggesting these pathways contribute to disease progression. ConclusionOur observations, together with the previously described associations between p53 signaling and both PI3K and HH pathway activity, provide evidence that increased PI3K and HH pathway activity and loss of ER pathway activity may be underlying events contributing to neoplastic transformation of FTE into STIC.

Role of EPA in Inflammation: Mechanisms, Effects, and Clinical Relevance.

Many chronic inflammatory processes are linked with the continuous release of inflammatory mediators and the activation of harmful signal-transduction pathways that are able to facilitate disease progression. In this context atherosclerosis represents the most common pathological substrate of coronary heart disease, and the characterization of the disease as a chronic low-grade inflammatory condition is now validated. The biomarkers of inflammation associated with clinical cardiovascular risk support the theory that targeted anti-inflammatory treatment appears to be a promising strategy in reducing residual cardiovascular risk. Several literature data highlight cardioprotective effects of the long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA). This PUFA lowers plasma triglyceride levels and has potential beneficial effects on atherosclerotic plaques. Preclinical studies reported that EPA reduces both pro-inflammatory cytokines and chemokines levels. Clinical studies in patients with coronary artery disease that receive pharmacological statin therapy suggest that EPA may decrease plaque vulnerability preventing plaque progression. This review aims to provide an overview of the links between inflammation and cardiovascular risk factors, importantly focusing on the role of diet, in particular examining the proposed role of EPA as well as the success or failure of standard pharmacological therapy for cardiovascular diseases.

Single-molecule view of slow and sequential conformational transitions of A2A adenosine receptor

G protein-coupled receptors (GPCRs) are expressed in all human tissues and have an important role in human physiology. The human genome encodes for more than 800 GPCRs that represent major targets for FDA-approved drugs. GPCRs are allosteric machines that bind many types of ligands on their extracellular surface and undergo conformational changes of the seven-transmembrane domain (7TM) that activate signal transduction pathways inside the cell. These ligand-dependent conformational dynamics and activation of receptors during signaling remain elusive.