Near-Infrared II Fluorescence Imaging and Image-Guided siRNA Therapy of Atherosclerosis.

Targeting Ca2+/calmodulin-dependent protein kinase γ (CaMKIIγ) in macrophages using RNAi nanotechnology represents an innovative and promising strategy in the diagnosis and treatment of atherosclerosis. Nevertheless, it remains elusive because of the current challenges associated with the systemic delivery of siRNA nanoparticle (NP) to atheromatous plaques and the complexity of atherosclerotic plaques. Here, we demonstrate the potential of a thienothiadiazole-based near-infrared-II (NIR-II) organic aggregation-induced emission (AIE) platform encapsulated with the Camk2g siRNA to effectively target CaMKIIγ in macrophages for dynamic imaging and image-guided gene therapy of atherosclerosis. The nanoparticles effectively decreased CaMKIIγ expression and increased the expression of the efferocytosis receptor MerTK in plaque macrophages, leading to a reduction in the necrotic core area of the lesion in an aortic plaque model. Our theranostic approach highlights the substantial promise of near-infrared II (NIR-II) AIEgens for imaging and image-guided therapy of atherosclerosis.

Targeting CAMK2N1/CAMK2 inhibits invasion, migration and angiogenesis of non-small cell lung cancer by promoting autophagy and apoptosis via AKT/mTOR signaling pathway.

Deregulation of calcium/calmodulin-dependent protein kinase II (CAMK2) inhibitor 1 (CAMK2N1) has been reported to be associated with the development of several malignancies. To date, there have been few studies on the role of CAMK2N1 in lung cancer. This study aimed to investigate the relationship between CAMK2N1 and the progression of non-small cell lung cancer (NSCLC). Methodological quality was assessed using the ARRIVE guidelines. CAMK2N1 was expressed at low levels in NSCLC tissues. Overexpression of CAMK2N1 in NSCLC cell lines resulted in changes such as proliferation inhibition, metastasis inhibition, autophagy increase, and apoptosis. Mechanistic studies revealed the regulatory role of CAMK2N1/CAMK2 in AKT/mTOR signaling. Upregulation of CAMK2N1 decreased the expression levels of phosphorylated calmodulin kinase 2 (p-CaMK2), phosphorylated Akt (p-Akt), and phosphorylated-mTOR (p-mTOR). In contrast, CAMK2 overexpression increased p-AKT and p-mTOR levels. Inhibition of autophagy or activation of AKT signaling reduced CAMK2N1-mediated tumor suppression. The tumorigenic ability of CAMK2N1 overexpressing cells significantly diminished in nude mice. In conclusion, this study demonstrated the cancer suppressive function of CAMK2N1 in NSCLC and showed that CAMK2N1/CAMK2 exerted anti-cancer effects by inhibiting the AKT/mTOR signaling pathway to promote autophagy.

The BK Channel Limits the Pro-Inflammatory Activity of Macrophages.

Macrophages play a crucial role in the innate immune response, serving as key effector cells in the defense against pathogens. Although the role of the large-conductance voltage and calcium-activated potassium channel, also known as the KCa1.1 or BK channel, in regulating neurotransmitter release and smooth muscle contraction is well known, its potential involvement in immune regulation remains unclear. We employed BK-knockout macrophages and noted that the absence of a BK channel promotes the polarization of macrophages towards a pro-inflammatory phenotype known as M1 macrophages. Specifically, the absence of the BK channel resulted in a significant increase in the secretion of the pro-inflammatory cytokine IL-6 and enhanced the activity of extracellular signal-regulated kinases 1 and 2 (Erk1/2 kinases), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and the transcription factor ATF-1 within M1 macrophages. Additionally, the lack of the BK channel promoted the activation of the AIM2 inflammasome without affecting the activation of the NLRC4 and NLRP3 inflammasomes. To further investigate the role of the BK channel in regulating AIM2 inflammasome activation, we utilized BK channel inhibitors, such as paxilline and iberiotoxin, along with the BK channel activator NS-11021. Pharmacological inactivation of the BK channel increased, and its stimulation inhibited IL-1ß production following AIM2 inflammasome activation in wild-type macrophages. Moreover, wild-type macrophages displayed increased calcium influx when activated with the AIM2 inflammasome, whereas BK-knockout macrophages did not due to the impaired extracellular calcium influx upon activation. Furthermore, under conditions of a calcium-free medium, IL-1ß production following AIM2 inflammasome activation was increased in both wild-type and BK-knockout macrophages. This suggests that the BK channel is required for the influx of extracellular calcium in macrophages, thus limiting AIM2 inflammasome activation. In summary, our study reveals a regulatory role of the BK channel in macrophages under inflammatory conditions.

TRIB2-Mediated Modulation of AMPK Promotes Hepatic Insulin Resistance.

Insulin resistance and its linked health complications are increasing in prevalence. Recent work has caused the role of Tribbles2 (TRIB2) in metabolism and cellular signaling to be increasingly appreciated, but its role in the progression of insulin resistance has not been elucidated. Here, we explore the functions of TRIB2 in modulating insulin resistance and the mechanism involved in insulin-resistant mice and palmitic acid-treated HepG2 cells. We demonstrate that whole-body knockout and hepatic-specific TRIB2 deficiency protect against diet-induced insulin resistance, inflammation, and endoplasmic reticulum stress. Accordingly, upregulation of TRIB2 in the liver aggravates these metabolic disturbances in high-fat diet-induced mice and ob/ob mice. Mechanistically, TRIB2 directly binds to the αγ-SBS domain of PRKAB through its pseudokinase domain, subsequently inhibiting the formation and activity of the AMPK complex. Moreover, the results of intervention against AMPK suggest that the effects of TRIB2 depend on AMPK. Our findings reveal that TRIB2 is a novel target for the treatment of insulin resistance and its associated metabolic complications and clarify the function of TRIB2 as a regulatory component of AMPK activity.

Shotgun Proteomic-Based Approach with a Q-Exactive Hybrid Quadrupole-Orbitrap High-Resolution Mass Spectrometer for Protein Adductomics on a 3D Human Brain Tumor Neurospheroid Culture Model: The Identification of Adduct Formation in Calmodulin-Dependent Protein Kinase-2 and Annexin-A1 Induced by Pesticide Mixture.

Pesticides are increasingly used in combinations in crop protection, resulting in enhanced toxicities for various organisms. Although protein adductomics is challenging, it remains a powerful bioanalytical tool to check environmental exposure and characterize xenobiotic adducts as putative toxicity biomarkers with high accuracy, facilitated by recent advances in proteomic methodologies and a mass spectrometry high-throughput technique. The present study aims to predict the potential neurotoxicity effect of imidacloprid and λ-cyhalothrin insecticides on human neural cells. Our protocol consisted first of 3D in vitro developing neurospheroids derived from human brain tumors and then treatment by pesticide mixture. Furthermore, we adopted a bottom-up proteomic-based approach using nanoflow ultraperformance liquid chromatography coupled with a high-resolution mass spectrometer for protein-adduct analysis with prediction of altered sites. Two proteins were selected, namely, calcium-calmodulin-dependent protein kinase-II (CaMK2) and annexin-A1 (ANXA1), as key targets endowed with primordial roles. De novo sequencing revealed several adduct formations in the active site of 82-ANXA1 and 228-CaMK2 as a result of neurotoxicity, predicted by the added mass shifts for the structure of electrophilic precursors. To the best of our knowledge, our study is the first to adopt a proteomic-based approach to investigate in depth pesticide molecular interactions and their potential to adduct proteins which play a crucial role in the neurotoxicity mechanism.

The Role of Death-Associated Protein Kinase-1 in Cell Homeostasis-Related Processes.

Tremendous amount of financial resources and manpower have been invested to understand the function of numerous genes that are deregulated during the carcinogenesis process, which can be targeted for anticancer therapeutic interventions. Death-associated protein kinase 1 (DAPK-1) is one of the genes that have shown potential as biomarkers for cancer treatment. It is a member of the kinase family, which also includes Death-associated protein kinase 2 (DAPK-2), Death-associated protein kinase 3 (DAPK-3), Death-associated protein kinase-related apoptosis-inducing kinase 1 (DRAK-1) and Death-associated protein kinase-related apoptosis-inducing kinase 2 (DRAK-2). DAPK-1 is a tumour-suppressor gene that is hypermethylated in most human cancers. Additionally, DAPK-1 regulates a number of cellular processes, including apoptosis, autophagy and the cell cycle. The molecular basis by which DAPK-1 induces these cell homeostasis-related processes for cancer prevention is less understood; hence, they need to be investigated. The purpose of this review is to discuss the current understanding of the mechanisms of DAPK-1 in cell homeostasis-related processes, especially apoptosis, autophagy and the cell cycle. It also explores how the expression of DAPK-1 affects carcinogenesis. Since deregulation of DAPK-1 is implicated in the pathogenesis of cancer, altering DAPK-1 expression or activity may be a promising therapeutic strategy against cancer.

TRIB2 safeguards naive T cell homeostasis during aging.

Naive CD4+ T cells are more resistant to age-related loss than naive CD8+ T cells, suggesting mechanisms that preferentially protect naive CD4+ T cells during aging. Here, we show that TRIB2 is more abundant in naive CD4+ than CD8+ T cells and counteracts quiescence exit by suppressing AKT activation. TRIB2 deficiency increases AKT activity and accelerates proliferation and differentiation in response to interleukin-7 (IL-7) in humans and during lymphopenia in mice. TRIB2 transcription is controlled by the lineage-determining transcription factors ThPOK and RUNX3. Ablation of Zbtb7b (encoding ThPOK) and Cbfb (obligatory RUNT cofactor) attenuates the difference in lymphopenia-induced proliferation between naive CD4+ and CD8+ cells. In older adults, ThPOK and TRIB2 expression wanes in naive CD4+ T cells, causing loss of naivety. These findings assign TRIB2 a key role in regulating T cell homeostasis and provide a model to explain the lesser resilience of CD8+ T cells to undergo changes with age.

Daclatasvir, an Antiviral Drug, Downregulates Tribbles 2 Pseudokinase and Resensitizes Enzalutamide-Resistant Prostate Cancer Cells.

FDA-approved enzalutamide is commonly prescribed to reduce the growth of advanced prostate cancer by blocking androgen receptor function. However, enzalutamide-resistant prostate cancer (ERPC) invariably develops and progresses to metastatic, lethal disease. Management of ERPC poses a special problem not only because available therapeutic regimens cannot effectively kill ERPC cells but also due to their propensity to invade large bones. Moreover, molecular mechanism(s) behind enzalutamide resistance is not properly understood, which is delaying development of newer agents. We found that the pseudokinase, Tribbles 2 (TRIB2), is overexpressed in ERPC cells and plays a critical role in their survival. Forced overexpression of TRIB2 enhances prostate cancer cell growth and confers resistance to physiologic doses of enzalutamide, suggesting that TRIB2 plays an important role in the development and progression of ERPC. Though TRIB2 has emerged as an excellent molecular target for ERPC, suitable inhibitors are not commercially available for effective targeting. By designing a luciferase-tagged TRIB2 fusion protein-based assay system, we screened a library of about 1,600 compounds and found that daclatasvir (DCV), an antiviral drug, effectively inhibits TRIB2-luciferase. We also found that DCV degrades TRIB2 proteins by direct binding and resensitizes ERPC cells to enzalutamide treatment. Moreover, DCV at lower, sublethal doses synergizes with enzalutamide to decrease the viability and induce apoptosis in prostate cancer cells. Because DCV is already approved by the FDA and well tolerated in humans, based on our findings, it appears that DCV is a promising new agent for development of an effective therapy for advanced, enzalutamide-resistant, lethal prostate cancer.

Uncovering perturbations in human hematopoiesis associated with healthy aging and myeloid malignancies at single-cell resolution.

Early hematopoiesis is a continuous process in which hematopoietic stem and progenitor cells (HSPCs) gradually differentiate toward specific lineages. Aging and myeloid malignant transformation are characterized by changes in the composition and regulation of HSPCs. In this study, we used single-cell RNA sequencing (scRNA-seq) to characterize an enriched population of human HSPCs obtained from young and elderly healthy individuals.Based on their transcriptional profile, we identified changes in the proportions of progenitor compartments during aging, and differences in their functionality, as evidenced by gene set enrichment analysis. Trajectory inference revealed that altered gene expression dynamics accompanied cell differentiation, which could explain aging-associated changes in hematopoiesis. Next, we focused on key regulators of transcription by constructing gene regulatory networks (GRNs) and detected regulons that were specifically active in elderly individuals. Using previous findings in healthy cells as a reference, we analyzed scRNA-seq data obtained from patients with myelodysplastic syndrome (MDS) and detected specific alterations of the expression dynamics of genes involved in erythroid differentiation in all patients with MDS such as TRIB2. In addition, the comparison between transcriptional programs and GRNs regulating normal HSPCs and MDS HSPCs allowed identification of regulons that were specifically active in MDS cases such as SMAD1, HOXA6, POU2F2, and RUNX1 suggesting a role of these transcription factors (TFs) in the pathogenesis of the disease.In summary, we demonstrate that the combination of single-cell technologies with computational analysis tools enable the study of a variety of cellular mechanisms involved in complex biological systems such as early hematopoiesis and can be used to dissect perturbed differentiation trajectories associated with perturbations such as aging and malignant transformation. Furthermore, the identification of abnormal regulatory mechanisms associated with myeloid malignancies could be exploited for personalized therapeutic approaches in individual patients.

Our blood contains many different types of cells; red blood cells carry oxygen through the body, platelets help to stop bleeding and a variety of white blood cells fight infections. All of these critical components come from a pool of immature cells in bone marrow, which can develop and specialise into any of these. However, as we get older, these immature cells can accumulate damage, including mutations in specific genes. This increases the risk of diseases such as myelodysplastic syndromes (MDS), a type of cancer in which the cells cannot develop and the patient does not have enough healthy mature blood cells. The changes in gene activity in the immature cells have previously been studied using samples from young and elderly people, as well as individuals with MDS. These studies examined large numbers of cells together, revealing differences between young and elderly people, and individuals with MDS. However, this does not describe how the different types alter their behaviour. To address this, Ainciburu, Ezponda et al. used a technique called single-cell RNA sequencing to study the gene activity in individual immature blood cells. This revealed changes associated with maturation that may account for the different combinations of cell populations in younger and older people. The results confirmed findings from previous studies and suggested new genes involved in ageing or MDS. Ainciburu, Ezponda et al. used these results to create an analytical system that highlights gene activity differences in individual MDS patients that are independent of age-related changes. These results provide new insights that could help further research into the development of MDS and the ageing process. In addition, scientists could study other diseases using this approach of analysing individual patients' gene activity. In future, this could help to personalise clinical decisions on diagnosis and treatment.

CaMK phosphatase (CaMKP/POPX2/PPM1F) inhibitors suppress the migration of human breast cancer MDA-MB-231 cells with loss of polarized morphology.

CaMK phosphatase (CaMKP/POPX2/PPM1F) is a Ser/Thr protein phosphatase that belongs to the PPM family. Accumulating evidence suggests that CaMKP is involved in the pathogenesis of various diseases, including cancer. To clarify the relationship between CaMKP activity and human breast cancer cell motility, we examined the phosphatase activity of CaMKP in cell extracts. CaMKP activity assays of the immunoprecipitates prepared from the cell extract revealed that cells exhibiting higher motility had higher CaMKP activity, with no significant differences in the specific activity being observed. Two CaMKP-specific inhibitors, 1-amino-8-naphthol-4-sulfonic acid (ANS) and 1-amino-8-naphthol-2,4-disulfonic acid (ANDS), inhibited the migration of highly invasive MDA-MB-231 breast cancer cells without significant cytotoxicity, while an inactive analog, naphthionic acid, did not. Furthermore, the cells lost their elongated morphology and assumed a rounded shape following treatment with ANS, whereas they retained their elongated morphology following treatment with naphthionic acid. Consistent with these findings, ANS and ANDS significantly enhanced the phosphorylation level of CaMKI, a cellular substrate of CaMKP, while naphthionic acid did not. The present data suggest that CaMKP could be a novel therapeutic target for cancer metastasis.

Tribbles Genes in Gastric Cancer: A Tumor-Suppressive Role for TRIB2.

Tribbles pseudokinases (TRIB1-3) are important signaling modulators involved in several cancers. However, their function in gastric cancer (GC) remains undefined. GC is still a deadly disease since the lack of sensitive and specific biomarkers for early diagnosis and therapy response prediction negatively affects patients' outcome. The identification of novel molecular players may lead to more effective diagnostic and therapeutic avenues. Therefore, we investigated the role of TRIB genes in gastric tumorigenesis. Data mining of the TCGA dataset revealed that chromosomal instability (CIN) tumors have lower TRIB2 and higher TRIB3 expression versus microsatellite instability (MSI)-high tumors, while TRIB1 levels are similar in both tumor types. Moreover, in CIN tumors, low TRIB2 expression is significantly associated with aggressive stage IV disease. As no studies on TRIB2 in GC are available, we focused on this gene for further in vitro analyses. We checked the effect of TRIB2 overexpression (OE) on MKN45 and NCI-N87 CIN GC cell lines. In MKN45 cells, TRIB2 OE reduced proliferation and colony formation ability and induced G2/M arrest, while it decreased the proliferation and cell motility of NCI-N87 cells. These effects were not mediated by the MAPK pathway. Our results suggest a tumor-suppressive function of TRIB2 in GC with a CIN phenotype.

Molecular Mechanisms Underlying Ca2+/Calmodulin-Dependent Protein Kinase Kinase Signal Transduction.

Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is the activating kinase for multiple downstream kinases, including CaM-kinase I (CaMKI), CaM-kinase IV (CaMKIV), protein kinase B (PKB/Akt), and 5'AMP-kinase (AMPK), through the phosphorylation of their activation-loop Thr residues in response to increasing the intracellular Ca2+ concentration, as CaMKK itself is a Ca2+/CaM-dependent enzyme. The CaMKK-mediated kinase cascade plays important roles in a number of Ca2+-dependent pathways, such as neuronal morphogenesis and plasticity, transcriptional activation, autophagy, and metabolic regulation, as well as in pathophysiological pathways, including cancer progression, metabolic syndrome, and mental disorders. This review focuses on the molecular mechanism underlying CaMKK-mediated signal transduction in normal and pathophysiological conditions. We summarize the current knowledge of the structural, functional, and physiological properties of the regulatory kinase, CaMKK, and the development and application of its pharmacological inhibitors.

A mutant-based analysis of the establishment of Nod-independent symbiosis in the legume Aeschynomene evenia.

Intensive research on nitrogen-fixing symbiosis in two model legumes has uncovered the molecular mechanisms, whereby rhizobial Nod factors activate a plant symbiotic signaling pathway that controls infection and nodule organogenesis. In contrast, the so-called Nod-independent symbiosis found between Aeschynomene evenia and photosynthetic bradyrhizobia, which does not involve Nod factor recognition nor infection thread formation, is less well known. To gain knowledge on how Nod-independent symbiosis is established, we conducted a phenotypic and molecular characterization of A. evenia lines carrying mutations in different nodulation genes. Besides investigating the effect of the mutations on rhizobial symbiosis, we examined their consequences on mycorrhizal symbiosis and in nonsymbiotic conditions. Analyzing allelic mutant series for AePOLLUX, Ca2+/calmodulin dependent kinase, AeCYCLOPS, nodulation signaling pathway 2 (AeNSP2), and nodule inception demonstrated that these genes intervene at several stages of intercellular infection and during bacterial accommodation. We provide evidence that AeNSP2 has an additional nitrogen-dependent regulatory function in the formation of axillary root hairs at lateral root bases, which are rhizobia-colonized infection sites. Our investigation of the recently discovered symbiotic actor cysteine-rich receptor-like kinase specified that it is not involved in mycorrhization; however, it is essential for both symbiotic signaling and early infection during nodulation. These findings provide important insights on the modus operandi of Nod-independent symbiosis and contribute to the general understanding of how rhizobial-legume symbioses are established by complementing the information acquired in model legumes.

Detecting endogenous TRIB2 protein expression by flow cytometry and Western blotting.

Protein kinases catalyze the transfer of a phosphate group thereby activating proteins and initiating signaling cascades. Their cousins, the pseudokinases, are enzymatically nonactive counterparts of protein kinases that can be considered zombie enzymes. Interestingly, pseudokinases, which constitute about 10% of the human kinome, have been implicated in many cancers, despite their sequences predicting a lack of catalytic activity. Owing to recent research, it has been demonstrated that dysregulation of many pseudokinases triggers changes in cell signaling, proliferation, and drug resistance. This review is aimed at describing methods that can be used for detection of Tribbles family of pseudokinases, specifically TRIB2. We describe intracellular staining by flow cytometry and Western blotting techniques for the detection of endogenous TRIB2 protein.

Analysis of human Tribbles 2 (TRIB2) pseudokinase.

Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a broad human protein interactome, including the well-studied AKT, C/EBPα and MAPK modules. Several lines of evidence indicate that human TRIB2 promotes cell survival and drug-resistance in solid tumors and blood cancers and is therefore of interest as a potential therapeutic target, although its physiological functions remain relatively poorly understood. The unique TRIB2 pseudokinase domain lacks the canonical 'DFG' motif, and subsequently possesses very low affinity for ATP in both the presence and absence of metal ions. However, TRIB2 also contains a unique cysteine-rich αC-helix, which interacts with a conserved peptide motif in its own carboxyl-terminal tail. This regulatory flanking region drives regulated interactions with distinct E3 ubiquitin ligases that serve to control the stability and turnover of TRIB2 client proteins. TRIB2 is also a low-affinity target of several known small-molecule protein kinase inhibitors, which were originally identified using purified recombinant TRIB2 proteins and a thermal shift assay. In this chapter, we discuss laboratory-based procedures for purification, stabilization and analysis of human TRIB2, including screening procedures that can be used for the identification of both reversible and covalent small molecule ligands.

TRIB2 regulates the expression of miR­33a­5p through the ERK/c­Fos pathway to affect the imatinib resistance of chronic myeloid leukemia cells.

Chronic myeloid leukemia (CML) is a hematological disease, and imatinib (IM) resistance represents a major problem for its clinical treatment. In the present study, the role of tribbles pseudokinase 2 (TRIB2) in IM resistance of CML and the possible mechanism were investigated. It was found that TRIB2 was highly expressed in IM­resistant patients with CML through the Oncomine database and this conclusion was confirmed using reverse transcription­quantitative PCR and western blot experiments. Knockdown of TRIB2 was found to increase the drug sensitivity of KG cells to IM using Cell­Counting Kit­8 (CCK­8) assays, and the low­expression TRIB2 mice were further found to be more sensitive to the IM and have a higher survival rate in leukemia model mice. Moreover, using western blot and luciferase experiments, it was found that TRIB2 could regulate c­Fos through the ERK signaling pathway, and c­Fos suppressed the transcriptional activity and the expression of miR­33a­5p. Further investigation identified that the binding site for c­Fos to function on miR­33a­5p was the ­958­965 region. Finally, CCK­8 assays and western blot experiments demonstrated that miR­33a­5p could inhibit the proliferation of KG cells and reduce IM resistance by suppressing the expression of HMGA2. In conclusion, it was demonstrated that TRIB2 regulates miR­33a­5p to reverse IM resistance in CML, which may help identify novel targets and therapeutic strategies for the clinical treatment of IM resistance.

Identification of a glioma functional network from gene fitness data using machine learning.

Glioblastoma multiforme (GBM) is an aggressive form of brain tumours that remains incurable despite recent advances in clinical treatments. Previous studies have focused on sub-categorizing patient samples based on clustering various transcriptomic data. While functional genomics data are rapidly accumulating, there exist opportunities to leverage these data to decipher glioma-associated biomarkers. We sought to implement a systematic approach to integrating data from high throughput CRISPR-Cas9 screening studies with machine learning algorithms to infer a glioma functional network. We demonstrated the network significantly enriched various biological pathways and may play roles in glioma tumorigenesis. From densely connected glioma functional modules, we further predicted 12 potential Wnt/ß-catenin signalling pathway targeted genes, including AARSD1, HOXB5, ITGA6, LRRC71, MED19, MED24, METTL11B, SMARCB1, SMARCE1, TAF6L, TENT5A and ZNF281. Cox regression modelling with these targets was significantly associated with glioma overall survival prognosis. Additionally, TRIB2 was identified as a glioma neoplastic cell marker in single-cell RNA-seq of GBM samples. This work establishes novel strategies for constructing functional networks to identify glioma biomarkers for the development of diagnosis and treatment in clinical practice.

Tribbles 2 pseudokinase confers enzalutamide resistance in prostate cancer by promoting lineage plasticity.

Enzalutamide, a second-generation antiandrogen, is commonly prescribed for the therapy of advanced prostate cancer, but enzalutamide-resistant, lethal, or incurable disease invariably develops. To understand the molecular mechanism(s) behind enzalutamide resistance, here, we comprehensively analyzed a range of prostate tumors and clinically relevant models by gene expression array, immunohistochemistry, and Western blot, which revealed that enzalutamide-resistant prostate cancer cells and tumors overexpress the pseudokinase, Tribbles 2 (TRIB2). Inhibition of TRIB2 decreases the viability of enzalutamide-resistant prostate cancer cells, suggesting a critical role of TRIB2 in these cells. Moreover, the overexpression of TRIB2 confers resistance in prostate cancer cells to clinically relevant doses of enzalutamide, and this resistance is lost upon inhibition of TRIB2. Interestingly, we found that TRIB2 downregulates the luminal markers androgen receptor and cytokeratin 8 in prostate cancer cells but upregulates the neuronal transcription factor BRN2 (Brain-2) and the stemness factor SOX2 (SRY-box 2) to induce neuroendocrine characteristics. Finally, we show that inhibition of either TRIB2 or its downstream targets, BRN2 or SOX2, resensitizes resistant prostate cancer cells to enzalutamide. Thus, TRIB2 emerges as a potential new regulator of transdifferentiation that confers enzalutamide resistance in prostate cancer cells via a mechanism involving increased cellular plasticity and lineage switching.

System analysis of VEGFA in renal cell carcinoma: The expression, prognosis, gene regulation network and regulation targets.

BACKGROUND: VEGFA is one of the most important regulators of angiogenesis and plays a crucial role in cancer angiogenesis and progression. Recent studies have highlighted a relationship between VEGFA expression and renal cell carcinoma occurrence. However, the expression level, gene regulation network, prognostic value, and target prediction of VEGFA in renal cell carcinoma remain unclear. Therefore, system analysis of the expression, gene regulation network, prognostic value, and target prediction of VEGFA in patients with renal cell carcinoma is of great theoretical significance as there is a clinical demand for the discovery of new renal cell carcinoma treatment targets and strategies to further improve renal cell carcinoma treatment efficacy. METHODS: This study used multiple free online databases, including cBioPortal, TRRUST, GeneMANIA, GEPIA, Metascape, UALCAN, LinkedOmics, Metascape, and TIMER for the abovementioned analysis. RESULTS: VEGFA was upregulated in patients with kidney renal clear cell carcinoma (KIRC) and kidney chromophobe (KICH), and downregulated in patients with kidney renal papillary cell carcinoma (KIRP). Moreover, genetic alterations of VEGFA were found in patients with renal cell carcinoma as follows: 4% (KIRC), 8% (KICH), and 4% (KIRP). The promoter methylation of VEGFA was lower and higher in patients with clinical stages of KIRC and stage 1 KIRP, respectively. VEGFA expression significantly correlated with KIRC and KIRP pathological stages. Furthermore, patients with KICH and KIRP having low VEGFA expression levels had a longer survival than those having high VEGFA expression levels. VEGFA and its neighboring genes functioned in the regulation of protein methylation and glycosylation, as well as muscle fiber growth and differentiation in patients with renal cell carcinoma. Gene Ontology enrichment analysis revealed that the functions of VEGFA and its neighboring genes in patients with renal cell carcinoma are mainly related to cell adhesion molecule binding, catalytic activity, acting on RNA, ATPase activity, actin filament binding, protease binding, transcription coactivator activity, cysteine-type peptidase activity, and calmodulin binding. Transcription factor targets of VEGFA and its neighboring genes in patients with renal cell carcinoma were found: HIF1A, TFAP2A, and ESR1 in KIRC; STAT3, NFKB1, and HIPK2 in KICH; and FOXO3, TFAP2A, and ETS1 in KIRP. We further explored the VEGFA-associated kinase (ATM in KICH as well as CDK1 and AURKB in KIRP) and VEGFA-associated microRNA (miRNA) targets (MIR-21 in KICH as well as MIR-213, MIR-383, and MIR-492 in KIRP). Furthermore, the following genes had the strongest correlation with VEGFA expression in patients with renal cell carcinoma: NOTCH4, GPR4, and TRIB2 in KIRC; CKMT2, RRAGD, and PPARGC1A in KICH; and FLT1, C6orf223, and ESM1 in KIRP. VEGFA expression in patients with renal cell carcinoma was positively associated with immune cell infiltration, including CD8+T cells, CD4+T cells, macrophages, neutrophils, and dendritic cells. CONCLUSIONS: This study revealed VEGFA expression and potential gene regulatory network in patients with renal cell carcinoma, thereby laying a foundation for further research on the role of VEGFA in renal cell carcinoma occurrence. Moreover, the study provides new renal cell carcinoma therapeutic targets and prognostic biomarkers as a reference for fundamental and clinical research.

Lithium modulates multiple tau kinases with distinct effects in cortical and hippocampal neurons according to concentration ranges.

The hyperphosphorylation of tau is a central mechanism in the pathogenesis of Alzheimer's disease (AD). Lithium is a potent inhibitor of glycogen synthase kinase-3beta (GSK3ß), the most important tau kinase in neurons, and may also affect tau phosphorylation by modifying the expression and/or activity of other kinases, such as protein kinase A (PKA), Akt (PKB), and calcium calmodulin kinase-II (CaMKII). The aim of the present study is to determine the effect of chronic lithium treatment on the protein expression of tau and its major kinases in cortical and hippocampal neurons, at distinct working concentrations. Primary cultures of cortical and hippocampal neurons were treated with sub-therapeutic (0.02 mM and 0.2 mM) and therapeutic (2 mM) concentrations of lithium for 7 days. Protein expression of tau and tau-kinases was determined by immunoblotting. An indirect estimate of GSK3ß activity was determined by the GSK3ß ratio (rGSKß). Statistically significant increments in the protein expression of tau and CaMKII were observed both in cortical and hippocampal neurons treated with subtherapeutic doses of lithium. GSK3ß activity was increased in cortical, but decreased in hippocampal neurons. Distinct patterns of changes in the expression of the remaining tau tau-kinases were observed: in cortical neurons, lithium treatment was associated with consistent decrements in Akt and PKA, whereas hippocampal neurons displayed increased protein expression of Akt and decreased PKA. Our results suggest that chronic lithium treatment may yield distinct biological effects depending on the concentration range, with regional specificity. We further suggest that hippocampal neurons may be more sensitive to the effect of lithium, presenting with changes in the expression of tau-related proteins at subtherapeutic doses, which may not be mirrored by the effects observed in cortical neurons.