Activation Of Signaling Proteins Research Articles

Exosomal miR-155-5p drives ibrutinib resistance in B-cell lymphoma

Ibrutinib, a Bruton Tyrosine Kinase (BTK) inhibitor, has shown effectiveness against various B-cell lymphoid malignancies. However, prolonged usage can induce resistance, affecting treatment outcomes. The oncogenic microRNA, miR-155-5p, is associated with poor prognosis in B-cell lymphomas, prompting our investigation into the mechanism of acquired ibrutinib resistance in these cells. We generated ibrutinib-resistant OCI-Ly1 cells (OCI-Ly1-IbtR) through continuous exposure to 1 μM and 2 μM of ibrutinib. We conducted microRNA profiling of OCI-Ly1-IbtR and isolated exosomes via ultracentrifugation. Comparative studies of microRNA levels in cells and exosomes, as well as exploration of targets of up-regulated microRNAs in OCI-Ly1-IbtR, were performed. Target validation involved transfection of candidate microRNAs, and co-culture experiments utilized OCI-Ly1 cells with exosomes from OCI-Ly1-IbtR. Elevated levels of miR-155-5p were observed in OCI-Ly1-IbtR and its exosomes, correlating with AKT and NF-κB activation. Transfection of miR-155-5p induced AKT/NF-κB pathway activation in OCI-Ly1, resulting in ibrutinib resistance, enhanced colony formation, and sustained BTK activity. Primary cell lines from ibrutinib-refractory B-cell lymphoma patients exhibited similar signaling protein activation. Target evaluation identified KDM5B and DEPTOR as miR-155-5p targets, confirmed by downregulation after transfection. We observed KDM5B and DEPTOR enrichment in Ago2 during ibrutinib resistance and miR-155-5p transfection. Co-culture experiments demonstrated exosome-mediated transfer of miR-155-5p, inducing ibrutinib resistance and KDM5B/DEPTOR downregulation in OCI-Ly1. Our findings suggest that miR-155-5p overexpression is associated with AKT and NF-κB pathway activation in ibrutinib-resistant cells, proposing a potential role for acquired miR-155-5p upregulation in B-cell lymphoma ibrutinib resistance.

Modification of Regulatory Tyrosine Residues Biases Human Hsp90α in its Interactions with Cochaperones and Clients

The highly conserved Hsp90 chaperones control stability and activity of many essential signaling and regulatory proteins including many protein kinases, E3 ligases and transcription factors. Thereby, Hsp90s couple cellular homeostasis of the proteome to cell fate decisions. High-throughput mass spectrometry revealed 178 and 169 posttranslational modifications (PTMs) for human cytosolic Hsp90α and Hsp90β, but for only a few of the modifications the physiological consequences are investigated in some detail. In this study, we explored the suitability of the yeast model system for the identification of key regulatory residues in human Hsp90α. Replacement of three tyrosine residues known to be phosphorylated by phosphomimetic glutamate and by non-phosphorylatable phenylalanine individually and in combination influenced yeast growth and the maturation of 7 different Hsp90 clients in distinct ways. Furthermore, wild-type and mutant Hsp90 differed in their ability to stabilize known clients when expressed in HepG2 HSP90AA1−/− cells. The purified mutant proteins differed in their interaction with the cochaperones Aha1, Cdc37, Hop and p23 and in their support of the maturation of glucocorticoid receptor ligand binding domain in vitro. In vivo and in vitro data correspond well to each other confirming that the yeast system is suitable for the identification of key regulatory sites in human Hsp90s. Our findings indicate that even closely related clients are affected differently by the amino acid replacements in the investigated positions, suggesting that PTMs could bias Hsp90s client specificity.

Copper doped carbon dots modified bacterial cellulose with enhanced antibacterial and immune regulatory functions for accelerating wound healing

The microenvironment of wound healing is susceptible to bacterial infection, chronic inflammation, oxidative stress, and inadequate angiogenesis, requiring the development of innovative wound dressings with antibacterial, anti-inflammatory, antioxidant, and angiogenic capabilities. This research crafted a new multifunctional bacterial cellulose composite membrane infused with copper-doped carbon dots (BC/Cu(II)-RCDs). Findings validated the successful loading of copper-doped carbon dots onto the BC membrane via hydrogen bonding interactions. Compared to the pure BC membrane, the BC/Cu(II)-RCDs composite membrane exhibited significantly enhanced hydrophilicity, tensile properties, and thermal stability. Diverse in vitro assays demonstrated excellent biocompatibility and antibacterial activity of BC/Cu(II)-RCDs composite membranes, alongside their ability to expedite the inflammatory phase and stimulate angiogenesis. In vivo trials corroborated the membrane's ability to foster epithelial regeneration, collagen deposition, and tissue regrowth in full-thickness skin wounds in rats while also curbing inflammation in infected full-thickness skin wounds. More importantly, the treatment of the BC/Cu(II)-RCDs composite membrane may result in the activation of VEGF and MAPK signaling proteins, which are key players in cell migration, angiogenesis, and skin tissue development. In essence, the developed BC/Cu(II)-RCDs composite membrane shows promise for treating infected wounds and serves as a viable alternative material for medicinal bandages.

A Comprehensive Systematic Review Coupled with an Interacting Network Analysis Identified Candidate Genes and Biological Pathways Related to Bovine Temperament.

Comprehension of the genetic basis of temperament has been improved by recent advances in the identification of genes and genetic variants. However, due to the complexity of the temperament traits, the elucidation of the genetic architecture of temperament is incomplete. A systematic review was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement to analyze candidate genes related to bovine temperament, using bovine as the population, SNPs and genes as the exposure, and temperament test as the outcome, as principal search terms for population, exposure, and outcome (PEO) categories to define the scope of the search. The search results allowed the selection of 36 articles after removing duplicates and filtering by relevance. One hundred-two candidate genes associated with temperament traits were identified. The genes were further analyzed to construct an interaction network using the STRING database, resulting in 113 nodes and 346 interactions and the identification of 31 new candidate genes for temperament. Notably, the main genes identified were SST and members of the Kelch family. The candidate genes displayed interactions with pathways associated with different functions such as AMPA receptors, hormones, neuronal maintenance, protein signaling, neuronal regulation, serotonin synthesis, splicing, and ubiquitination activities. These new findings demonstrate the complexity of interconnected biological processes that regulate behavior and stress response in mammals. This insight now enables our targeted analysis of these newly identified temperament candidate genes in bovines.

Comprehensive Transcriptome and Proteome Analyses Reveal the Drought Responsive Gene Network in Potato Roots.

The root system plays a decisive role in the growth and development of plants. The water requirement of a root system depends strongly on the plant species. Potatoes are an important food and vegetable crop grown worldwide, especially under irrigation in arid and semi-arid regions. However, the expected impact of global warming on potato yields calls for an investigation of genes related to root development and drought resistance signaling pathways in potatoes. In this study, we investigated the molecular mechanisms of different drought-tolerant potato root systems in response to drought stress under controlled water conditions, using potato as a model. We analyzed the transcriptome and proteome of the drought-sensitive potato cultivar Atlantic (Atl) and the drought-tolerant cultivar Qingshu 9 (Q9) under normal irrigation (CK) and weekly drought stress (D). The results showed that a total of 14,113 differentially expressed genes (DEGs) and 5596 differentially expressed proteins (DEPs) were identified in the cultivars. A heat map analysis of DEGs and DEPs showed that the same genes and proteins in Atl and Q9 exhibited different expression patterns under drought stress. Weighted gene correlation network analysis (WGCNA) showed that in Atl, Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG)-enriched pathways were related to pyruvate metabolism and glycolysis, as well as cellular signaling and ion transmembrane transporter protein activity. However, GO terms and KEGG-enriched pathways related to phytohormone signaling and the tricarboxylic acid cycle were predominantly enriched in Q9. The present study provides a unique genetic resource to effectively explore the functional genes and uncover the molecular regulatory mechanism of the potato root system in response to drought stress.

NAD activates olfactory receptor 1386 to regulate type I interferon responses in Plasmodium yoelii YM infection

Olfactory receptors (Olfr) are G protein-coupled receptors that are normally expressed on olfactory sensory neurons to detect volatile chemicals or odorants. Interestingly, many Olfrs are also expressed in diverse tissues and function in cell-cell recognition, migration, and proliferation as well as immune responses and disease processes. Here, we showed that many Olfr genes were expressed in the mouse spleen, linked to Plasmodium yoelii genetic loci significantly, and/or had genome-wide patterns of LOD scores (GPLSs) similar to those of host Toll-like receptor genes. Expression of specific Olfr genes such as Olfr1386 in HEK293T cells significantly increased luciferase signals driven by IFN-β and NF-κB promoters, with elevated levels of phosphorylated TBK1, IRF3, P38, and JNK. Mice without Olfr1386 were generated using the CRISPR/Cas9 method, and the Olfr1386-/- mice showed significantly lower IFN-α/β levels and longer survival than wild-type (WT) littermates after infection with P. yoelii YM parasites. Inhibition of G protein signaling and P38 activity could affect cyclic AMP-responsive element promoter-driven luciferase signals and IFN-β mRNA levels in HEK293T cells expressing the Olfr1386 gene, respectively. Screening of malaria parasite metabolites identified nicotinamide adenine dinucleotide (NAD) as a potential ligand for Olfr1386, and NAD could stimulate IFN-β responses and phosphorylation of TBK1 and STAT1/2 in RAW264.7 cells. Additionally, parasite RNA (pRNA) could significantly increase Olfr1386 mRNA levels. This study links multiple Olfrs to host immune response pathways, identifies a candidate ligand for Olfr1386, and demonstrates the important roles of Olfr1386 in regulating type I interferon (IFN-I) responses during malaria parasite infections.

The interplay between membrane viscosity and ligand-binding receptor kinetics in lipid bilayers

Plasma membranes appear as deformable systems wherein molecules are free to move and diffuse giving rise to condensed microdomains (composed of ordered lipids, transmembrane proteins and cholesterol) surrounded by disordered lipid molecules. Such denser and thicker regions, namely lipid rafts, are important communication hubs for cells. Indeed, recent experiments revealed how the most of active signaling proteins co-localize on such domains, thereby intensifying the biochemical trafficking of substances. From a material standpoint, it is reasonable to assume the bilayer as a visco-elastic body accounting for both in-plane fluidity and elasticity. Consequently, lipid rafts contribute to membrane heterogeneity by typically exhibiting higher stiffness and viscosity and by locally altering the bilayer dynamics and proteins activity. A chemo-mechanical model of lipid bilayer coupled with interspecific dynamics among the resident species (typically transmembrane receptors and trasporters) has been recently formulated to explain and predict how proteins regulate the dynamic heterogeneity of membrane. However, the explicit inclusion of the membrane viscosity in the model was not considered. To this aim, the present work enriches the constitutive description of the bilayer by modeling its visco-elastic behavior. This is done through a strain-level dependent viscosity able to theoretically trace back the alteration of membrane fluidity experimentally observed in lipid phase transitions. This provides new insights into how the quasi-solid and fluid components of lipid membrane response interact with the evolution of resident proteins by affecting the activity of raft domains, with effects on cell mechano-signaling.

Atypical chemokine receptor 4 (ACKR4/CCX-CKR): A comprehensive exploration across physiological and pathological landscapes in contemporary research.

Atypical chemokine receptor 4 (ACKR4), also known as CCX-CKR, is a member of the chemokine receptor family that lacks typical G protein signaling activity. Instead, ACKR4 functions as a scavenger receptor that can bind and internalize a wide range of chemokines, influencing their availability and activity in the body. ACKR4 is involved in various physiological processes, such as immune cell trafficking and the development of thymus, spleen, and lymph nodes. Moreover, ACKR4 has been implicated in several pathological conditions, including cancer, heart and lung diseases. In cancer, ACKR4 plays a complex role, acting as a tumor suppressor or promoter depending on the type of cancer and the stage of the disease. For instance, ACKR4 may inhibit the growth and metastasis of breast cancer, but it may also promote the progression of hepatocellular carcinoma and gastric cancer. In inflammatory situations, ACKR4 has been found to modulate the recruitment and activation of immune cells, contributing to the pathogenesis of diseases such as myocardial infraction and pulmonary sarcoidosis. The study of ACKR4 is still ongoing, and further research is needed to fully understand its role in different physiological and pathological contexts. Nonetheless, ACKR4 represents a promising target for the development of novel therapeutic strategies for various diseases.

Computationally designed sensors detect endogenous Ras activity and signaling effectors at subcellular resolution.

The utility of genetically encoded biosensors for sensing the activity of signaling proteins has been hampered by a lack of strategies for matching sensor sensitivity to the physiological concentration range of the target. Here we used computational protein design to generate intracellular sensors of Ras activity (LOCKR-based Sensor for Ras activity (Ras-LOCKR-S)) and proximity labelers of the Ras signaling environment (LOCKR-based, Ras activity-dependent Proximity Labeler (Ras-LOCKR-PL)). These tools allow the detection of endogenous Ras activity and labeling of the surrounding environment at subcellular resolution. Using these sensors in human cancer cell lines, we identified Ras-interacting proteins in oncogenic EML4-Alk granules and found that Src-Associated in Mitosis 68-kDa (SAM68) protein specifically enhances Ras activity in the granules. The ability to subcellularly localize endogenous Ras activity should deepen our understanding of Ras function in health and disease and may suggest potential therapeutic strategies.

Overexpression and Purification of Mitogenic and Metabolic Fibroblast Growth Factors.

Fibroblast growth factors (FGFs) are proteins with a vast array of biological activity, such as cell development and repair, glucose and bile acid metabolisms, and wound healing. Due to their critical and diverse physiological functions, FGFs are believed to possess potential as therapeutic agents for many diseases and conditions that warrant further investigations. Thus, a simple, cost-efficient method to purify these biologically active signaling proteins is desirable. Herein, we introduce such techniques to purify FGFs that possess either high heparin-binding affinity or low to no heparin-binding affinity. This method takes advantage of the high affinity toward heparin sulfate from paracrine FGF1 to isolate the targeted protein. It also accounts for FGF members that have low heparin affinity, such as the metabolic FGFs, by introducing poly-histidine tags in the recombinant protein in combination with the immobilized metal affinity chromatography. Subsequently, the purified FGF products are separated from the other small protein by high-speed centrifugation. Products are then subjected to other biophysical experiments like SDS-PAGE, mass spectrometry, circular dichroism, intrinsic fluorescence, isothermal titration calorimetry, differential scanning calorimetry, and biological cell activity assay to confirm that the target proteins are purified with intact native conformation and no significant change in the intrinsic characteristics and biological activities.

S-glutathionylation and S-nitrosylation as modulators of redox-dependent processes in cancer cell

The development of oxidative/nitrosative stress associated with the activation of oncogenic pathways is a consequence of an increase in the level of generation of reactive oxygen and nitrogen species (ROS/RNS) in tumor cells. The action of ROS/RNS is dual: high levels cause cell death and limit tumor growth at certain phases of malignant neoplasm development, while low concentrations allow oxidative/nitrosative modifications of key redox-dependent residues in regulatory proteins. The reversibility of such modifications as S-glutathionylation/S-nitrosylation, which occur through the electrophilic attack of ROS/RNS on the nucleophilic residues of Cys, provides redox switching in the activity of signaling proteins and the ability to control the processes of proliferation and programmed death. The level of S-glutathionylated and S-nitrosylated proteins is controlled by the balance between S-glutathionylation/deglutathionylation and S-nitrosylation/denitrosylation, the ratio of which depends on the cellular redox status. The degree of S-glutathionylation and S-nitrosylation of protein targets and their ratio largely determines the state and directions of signaling pathways in cancer cells. The review discusses the features of S-glutathionylation and S-nitrosylation reactions in cancer cells, the balance of systems that control their activity and their relationship with redox-dependent processes and tumor growth.

Phenomenon of suppression of estrogen signaling in breast cancer cells under ultraviolet irradiation: role of Snail proteins

Introduction. The study of the effect of irradiation or any other DNA-damaging agents on the sensitivity of tumors to conservative therapy, drug or hormonal, is among the most imporant tasks that determine the efficiency of combined therapy of cancer patients.Aim. To investigate the effect of irradiation on the activity of key signaling proteins and the level of hormone dependence of breast cancer cells.Materials and methods. The experiments were performed on in vitro cultured estrogen-dependent MCF-7 breast cancer cells. Ultraviolet (UV) irradiation in the range of 254 nm with the intensity of 25–50 J/m2 was used as an experimental model to study the response of tumor cells to DNA damage. Cell growth rate was determined using the MTT test, cell survival after irradiation was analyzed using the colony-forming test. Estrogen receptor transcriptional activity was determined by reporter assay; cellular protein expression was determined by immunoblotting.Results. Single UV irradiation of MCF-7 cells leads to a marked increase in the level of apoptotic markers: p53, cPARP, suppression of expression of growth signaling proteins: CDK4/6 and estrogen receptor α (ERα). The above changes are accompanied with an increase in phosphorylation of Akt protein kinase and a marked increase in the expression of Snail1, one of the key proteins of epithelial-mesenchymal transition. In UV-resistant MCF-7/UVR cell subline obtained under repeated irradiation cycles, the levels of apoptotic and growth signaling proteins (p53, cPARP, CDK4/6) return to control levels, except for the phosphorylated form of Akt and Snail1, whose content remains high. Transfection of Snail1-expressing plasmid into MCF-7 cells is accompanied by activation of apoptotic signaling, suppression of ERα activity, and development of partial hormone resistance; however, the sensitivity of cells to irradiation is practically unchanged. Transfection of microRNA-181a-2, one of the microRNAs associated with cell resistance, simultaneously activates Akt and Snail1 and leads to the development of cross-resistance of cells to irradiation and hormonal drugs.Conclusion. The obtained data allow us to consider irradiation-induced Snail1 activation as one of the factors involved in deregulation of estrogen signaling and formation of cell resistance to hormonal drugs, while simultaneous activation of Akt and Snail1 is accompanied by the development of cross-resistance to irradiation and hormonal drugs.

The Uses of Anabolic Androgenic Steroids Among Athletes; Its Positive and Negative Aspects- A Literature Review.

The use of anabolic androgenic steroids (AAS) for strength training and muscle building is a widespread practice among athletes and young individuals. Athletes and bodybuilders are using these substances for various purposes, such as enhancing muscle mass, strengthening their bodies, and enhancing their performances. AAS exert a wide range of physiological effects that result in the activation of central signaling, resulting in adverse effects. Moreover, excessive use of AAS which can be categorized as AAS abuse; is linked to biological and psychological pathologies, which can lead to mortality. Complications arising from steroid abuse involve both cellular and physiological complications. Cellular complications arise when activation of signaling proteins like mTOR, Akt, etc. leads to alteration in protein synthesis pathways, cell cycle, oxidative stress, and apoptosis, contributing to damage at the cellular level. Physiological complications are evident with cardiovascular pathologies, including an altered lipid profile, cardiac hypertrophy, hypogonadism after discontinuation of AAS, and modulation of GABA receptors in the brain, all contributed by the androgen receptor signaling. Clinical complications budding from these altered physiological processes lead to clinical effects like testicular dysfunction, acne, gynecomastia, and neuropsychiatric disorders. Despite potential therapeutic benefits, AAS use is prohibited by the World Anti-Doping Agency (WADA) due to concerns over adverse health effects. This review highlights the molecular mechanisms, physiological processes, and clinical complications arising from the excessive use of AAS among athletes.

Abstract B025: The next generation farnesyltransferase inhibitor, KO-2806, blocks oncogenic signaling at multiple nodes to enhance the antitumor efficacy of KRASG12C inhibitor adagrasib in KRASG12C non-small cell lung carcinoma

Abstract Emerging clinical and preclinical data have shown that responses to KRASG12C inhibition are limited by the activation of compensatory signaling proteins, including receptor tyrosine kinases (RTKs) and mTOR. Combination therapeutic strategies co-targeting these nodes are needed to fully realize the potential of breakthrough KRASG12C-selective inhibitors. We propose that, given its multipronged mechanism of action, KO-2806, a next-generation farnesyltransferase inhibitor (FTI) with increased potency and improved pharmacokinetic properties relative to earlier FTI candidates that is poised to enter a first-in-human clinical trial, is a uniquely suited combination partner for KRASG12C inhibitors. Here, we evaluate the therapeutic potential of combined KO-2806 and adagrasib in preclinical models of KRASG12C non-small cell lung cancer (NSCLC). Our previous work indicated that FTIs amplify the antitumor effects of PI3Kα inhibition by blocking compensatory mTOR signaling. As activation of compensatory signaling is a well-described bypass mechanism to escape KRAS inhibition, we asked whether KO-2806 could improve the in vivo efficacy of adagrasib. Combination of KO-2806 with adagrasib led to markedly enhanced antitumor effects in KRASG12C-mutant NSCLC cell line- and patient-derived xenograft models compared to adagrasib alone. Tumor regression in the combination group corresponded to increased apoptosis, as evidenced by higher cleaved caspase 3 staining in combination-treated tumors compared to single agent groups. Intriguingly, adagrasib induced expression of the RTK HER3, a potential driver of bypass signaling, while HER3 expression in combination-treated tumors was lower than in vehicle-treated tumors. To further interrogate the mechanistic basis of the observed in vivo synergy, we cultured KRASG12C-mutant cell lines as 3D tumor spheroids and exposed them to KO-2806 and/or adagrasib. Consistent with the xenograft tumor data, spheroids exposed to the combination expressed lower levels of the upstream receptor HER3 (total and phosphorylated forms) and higher levels of apoptotic markers cleaved PARP and cleaved caspase 3. Concurrently, activity of the downstream effectors ERK and mTOR was reduced. Co-exposure to KO-2806 and adagrasib potently inhibited phosphorylation of ERK and its substrate p90 RSK as well as phosphorylation of mTOR substrates p70 S6K, 4EBP1, and S6. The mTOR signaling reduction paralleled defarnesylation of the farnesylation-dependent mTOR activator RHEB, so we depleted RHEB expression in spheroids and assayed their growth. Genetic depletion of RHEB phenocopied the antiproliferative effects of KO-2806 in combination with adagrasib, implicating RHEB as an important farnesylated target in KRASG12C NSCLC. These preclinical results suggest that KO-2806 is an effective combination partner for KRASG12C inhibitors by simultaneously targeting an upstream activator (HER3) and a downstream effector (RHEB/mTOR) of KRAS, leading to improved antitumor activity in KRASG12C-mutant NSCLC models. Citation Format: Hetika V Patel, Alison Smith, Stacia Chan, Linda Kessler, Francis Burrows, Shivani Malik. The next generation farnesyltransferase inhibitor, KO-2806, blocks oncogenic signaling at multiple nodes to enhance the antitumor efficacy of KRASG12C inhibitor adagrasib in KRASG12C non-small cell lung carcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B025.

Protein signaling and drug target activation signatures to guide therapy prioritization: Therapeutic resistance and sensitivity in the I-SPY 2 Trial

Molecular subtyping of breast cancer is based mostly on HR/HER2 and gene expression-based immune, DNA repair deficiency, and luminal signatures. We extend this description via functional protein pathway activation mapping using pre-treatment, quantitative expression data from 139 proteins/phosphoproteins from 736 patients across 8 treatment arms of the I-SPY 2 Trial (ClinicalTrials.gov: NCT01042379). We identify predictive fit-for-purpose, mechanism-of-action-based signatures and individual predictive protein biomarker candidates by evaluating associations with pathologic complete response. Elevated levels of cyclin D1, estrogen receptor alpha, and androgen receptor S650 associate with non-response and are biomarkers for global resistance. We uncover protein/phosphoprotein-based signatures that can be utilized both for molecularly rationalized therapeutic selection and for response prediction. We introduce a dichotomous HER2 activation response predictive signature for stratifying triple-negative breast cancer patients to either HER2 or immune checkpoint therapy response as a model for how protein activation signatures provide a different lens to view the molecular landscape of breast cancer and synergize with transcriptomic-defined signatures.

Co-targeting FAK and Gli1 inhibits the tumor-associated macrophages-released CCL22-mediated esophageal squamous cell carcinoma malignancy.

Esophageal squamous cell carcinoma (ESCC) is a frequently seen esophageal tumor type in China. Activation of signaling proteins and relevant molecular mechanisms in ESCC are partially explored, impairing the antitumor efficiency of targeted therapy in ESCC treatment. Tumor-associated macrophages (TAMs)-released C-C motif chemokine 22 (CCL22) can activate intratumoral focal adhesion kinase (FAK), thus promoting the progression of ESCC. Here, we demonstrated that highly secreted CCL22 by TAMs (CCL22-positive TAMs) induced ESCC cell stemness and invasion through facilitating transcriptional activity of intratumoral glioma-associated oncogene 1 (Gli1), a downstream effector for Hedgehog (HH) pathway. Mechanistically, FAK-activated protein kinase B (AKT) mediated Gli1 phosphorylation at its Ser112/Thr115/Ser116 sites and released Gli1 from suppressor of fused homolog, the endogenous inhibitor of Gli1 to activate downstream stemness-associated factors, such as SRY-box transcription factor 2 (SOX2), Nanog homeobox (Nanog), or POU class 5 homeobox (OCT4). Furthermore, inhibition of FAK activity by VS-4718, the FAK inhibitor, enhanced antitumor effect of GDC-0449, the HH inhibitor, both in xenografted models and in vitro assays. Clinically, CCL22/Gli1 axis is used to evaluate ESCC prognosis. Overall, our study establishes the communication of FAK with HH pathway and offers the novel mechanism related to Gli1 activation independent of Smoothened as well as the rationale for the anti-ESCC combination treatment.

The tumor suppressive effect and apoptotic mechanism of TRAIL gene-containing recombinant NDV in TRAIL-resistant colorectal cancer HT-29 cells and TRAIL-nonresistant HCT116 cells, with each cell bearing a mouse model.

TRAIL is an anticancer drug that induces cancer cell apoptosis by interacting with death receptors (DRs). However, owing to low cell-surface expression of DRs, certain colorectal cancer (CRC) cells resist TRAIL-induced apoptosis. Newcastle disease virus (NDV) infection can elevate DR protein expression in cancer cells, potentially influencing their TRAIL sensitivity. However, the precise mechanism by which NDV infection modulates DR expression and impacts TRAIL sensitivity in cancer cells remains unknown. Herein, we developed nonpathogenic NDV VG/GA strain-based recombinant NDV (rNDV) and TRAIL gene-containing rNDV (rNDV-TRAIL). We observed that viral infections lead to increased DR and TRAIL expressions and activate signaling proteins involved in intrinsic and extrinsic apoptosis pathways. Experiments were conducted invitro using TRAIL-resistant CRC cells (HT-29) and nonresistant CRC cells (HCT116) and invivo using relevant mouse models. rNDV-TRAIL was found to exhibit better apoptotic efficacy than rNDV in CRC cells. Notably, rNDV-TRAIL had the stronger cancer cell-killing effect in TRAIL-resistant CRC cells. Western blot analyses showed that both rNDV and rNDV-TRAIL infections activate signaling proteins involved in the intrinsic and extrinsic apoptotic pathways. Notably, rNDV-TRAIL promotes concurrent intrinsic and extrinsic signal transduction in both HCT-116 and HT-29 cells. Therefore, rNDV-TRAIL infection effectively enhances DR expression in DR-depressed HT-29 cells. Moreover, the TRAIL protein expressed by rNDV-TRAIL effectively interacts with DR, leading to enhanced apoptosis in TRAIL-resistant HT-29 cells. Therefore, rNDV-TRAIL has potential as a promising therapeutic approach for treating TRAIL-resistant cancers.

Anthracycline cardiotoxicity is exacerbated by global p38b genetic ablation in a sexually dimorphic manner but unaltered by cardiomyocyte-specific p38a loss.

Severe cardiotoxic effects limit the efficacy of doxorubicin (DOX) as a chemotherapeutic agent. Activation of intracellular stress signaling networks, including p38 mitogen-activated protein kinase (MAPK), have been implicated in DOX-induced cardiotoxicity (DIC). However, the roles of the individual p38 isoforms in DIC remain incompletely elucidated. We recently reported that global p38d deletion protected female but not male mice from DIC, while global p38g deletion did not significantly modulate it. Here we studied the in vivo roles of p38a and p38b in acute DIC. Male and female mice with cardiomyocyte-specific deletion of p38a or global deletion of p38b and their wild-type counterparts were injected with DOX. Survival and health were tracked for 10 days post-injection. Cardiac function was assessed by echocardiography and electrocardiography, and fibrosis by Picro Sirius Red staining. Expression and activation of signaling proteins and inflammatory markers were measured by western blot, phosphorylation array and chemokine/cytokine array, respectively. Global p38b deletion significantly aggravated DIC and worsened cardiac electrical and mechanical function deterioration in female mice. Mechanistically, DIC in p38b-null female mice correlated with increased autophagy, sustained hyperactivation of pro-apoptotic JNK signaling, as well as remodeling of myocardial inflammatory environment. In contrast, cardiomyocyte-specific deletion of p38a improved survival of DOX30-treated male mice 5 days post-treatment but did not influence cardiac function in DOX-treated male or female mice. Our data highlight the sex- and isoform-specific roles of p38a and p38b MAPKs in DOX-induced cardiac injury and suggest a novel in vivo function of p38b in protecting female mice from DIC.

Development of a membrane-based Gi-CASE biosensor assay for profiling compounds at cannabinoid receptors.

Introduction: The cannabinoid receptor (CBR) subtypes 1 (CB1R) and 2 (CB2R) are key components of the endocannabinoid system (ECS), playing a central role in the control of peripheral pain, inflammation and the immune response, with further roles in the endocrine regulation of food intake and energy balance. So far, few medicines targeting these receptors have reached the clinic, suggesting that a better understanding of the receptor signalling properties of existing tool compounds and clinical candidates may open the door to the development of more effective and safer treatments. Both CB1R and CB2R are Gαi protein-coupled receptors but detecting Gαi protein signalling activity reliably and reproducibly is challenging. This is due to the inherent variability in live cell-based assays and restrictions around the use of radioactive [35S]-GTPγS, a favoured technology for developing higher-throughput membrane-based Gαi protein activity assays. Methods: Here, we describe the development of a membrane-based Gαi signalling system, produced from membrane preparations of HEK293TR cells, stably overexpressing CB1R or CB2R, and components of the Gαi-CASE biosensor. This BRET-based system allows direct detection of Gαi signalling in both cells and membranes by monitoring bioluminescence resonance energy transfer (BRET) between the α and the βγ subunits. Cells and membranes were subject to increasing concentrations of reference cannabinoid compounds, with 10μM furimazine added to generate RET signals, which were detected on a PHERAstar FSX plate reader, then processed using MARS software and analysed in GraphPad PRISM 9.2. Results: In membranes expressing the Gi-CASE biosensor, the cannabinoid ligands profiled were found to show agonist and inverse agonist activity. Agonist activity elicited a decrease in the BRET signal, indicative of receptor activation and G protein dissociation. Inverse agonist activity caused an increase in BRET signal, indicative of receptor inactivation, and the accumulation of inactive G protein. Our membrane-based Gi-CASE NanoBRET system successfully characterised the potency (pEC50) and efficacy (Emax) of CBR agonists and inverse agonists in a 384-well screening format. Values obtained were in-line with whole-cell Gi-CASE assays and consistent with literature values obtained in the GTPγS screening format. Discussion: This novel, membrane-based Gαi protein activation assay is applicable to other Gαi-coupled GPCRs, including orphan receptors, allowing real-time higher-throughput measurements of receptor activation.

Bioinformatics Analysis of the Genetic and Epigenetic Alterations of Bone Morphogenetic Protein Receptors in Metastatic Breast Cancer.

The leading cause of mortality in patients with breast cancer is metastasis, and bone morphogenetic protein (BMP) signaling activation regulates metastasis in breast cancer. This study explored the genetic and epigenetic modification of BMP receptor genes associated with metastatic breast cancer cells using bioinformatics. The genetic and epigenetic alterations of BMP receptors (BMPR1A, BMPR1B, BMPR2, ACVR2A, ACVR1, ACVR2B, ACVR1B, HJV, and ENG) were examined using cBioportal and methSurv, respectively. mRNA expression was analyzed using TNM plot and bcgenex, and protein expression was studied using Human Protein Atlas. Prognostic value and ROC were investigated using Kaplan-Meier (KM) and ROC plot, respectively. Finally, mutant function was predicted using several databases, including PolyPhen-2, FATHMM, Mutation Assessor, and PredictSNP. Oncoprint analysis showed genetic alterations in BMPR1A (39%), BMPR1B (13%), BMPR2 (34%), ACVR2A (14%), ACVR1 (7%), ACVR2B (13), ACVR1B (35%), HJV (40%), and ENG (33%) across the patients with breast cancer in The Metastatic Breast Cancer Project. The mRNA and protein levels of BMPR2 were increased in metastatic breast tumor tissues compared with those in normal and breast tumor tissues. BMPR1A and BMPR2 showed the highest and lowest levels of epigenetic alterations among the BMP receptors, respectively. The patients with breast cancer who had low levels of BMPR2 had a better overall survival (OS) than those with high levels of BMPR2. Functional mutation prediction showed that mutants in BMPR2 (R272L, E274K, and L685F), ACVR2A (S127L), and ACVR1B (R484H), are deleterious, probably damaging, and possess a cancer phenotype. ROC plot revealed no BMP receptors correlated with endocrine therapy sensitivity. BMPR1B, BMPR2, and ACVR2A levels were significantly linked as moderate prediction of anti-HER2, BMPR2, and ACVR1B demonstrated moderate predictive potential for chemotherapy sensitivity. This study contributed in fully comprehending the significance of genetic and epigenetic alterations in BMP receptors and BMP signaling in metastatic breast cancer cells for the development of breast cancer treatment plans.