Cell and Bioscience最新文献

N-myc downstream-regulated gene 1 (NDRG1) is a member of the NDRG family of intracellular proteins and plays a central role in a wide range of biological processes including stress response, differentiation, and metabolism. The overexpression of NDRG1 is an indicator of poor prognosis in various types of cancer. Here, we found that NDRG1 is an independent prognostic marker of poor outcome in breast cancer (BC). Analysis of the TCGA dataset showed a significant positive correlation between NDRG1 and PRKCA expression, suggesting a mechanistic role of protein kinase C (PKC) in the regulation of NDRG1. We then assessed the hypothesis that PKC might modulate the activity of NDRG1, and observed that different acute stress conditions converging on PKC activation lead to enhanced NDRG1 expression. This mechanism was found to be specific for NDRG1 as the expression of other NDRG members was not affected. Moreover, CRISPR-based inhibition of NDRG1 expression was obtained in a BC cell line, and showed that this protein is a key driver of BC cell invasion through the Rho-associated coiled-coil containing protein kinase 1 (ROCK1)/phosphorylated cofilin pathway that regulates stress fiber assembly, and the modulation of extracellular matrix reorganization related genes. Together, our findings highlight the potential of NDRG1 as a new BC biomarker and uncover a novel mechanism of regulation of NDRG1 expression that might lead to innovative therapeutic strategies.

Background: We have previously developed a candidate therapeutic HPV DNA vaccine (pBI-11) encoding mycobacteria heat shock protein 70 linked to HPV16/18 E6/E7 proteins for the control of advanced HPV-associated oropharyngeal cancer (NCT05799144). While naked DNA vaccines are readily produced, stable, and well tolerated, their potency is limited by the delivery efficiency. Here we compared three different IM delivery strategies, including intramuscular (IM) injection, either with a needle alone or with electroporation at the injection site, and a needle-free injection system (NFIS), for their ability to elicit gene expression and to improve the potency of pBI-11 DNA vaccine.

Results: We found that electroporation after IM injection significantly increases gene expression from a luciferase-encoding DNA construct compared to IM injection alone or NFIS. We also showed that single administration of pBI-11 DNA via electroporation-mediated delivery generates the greatest increase in HPV antigen-specific CD8 + T cell-mediated immune responses, resulting in the most potent antitumor effect compared to the other two methods. We further compared the response to three repeat immunizations via each of these different methods. We found that electroporation-mediated delivery of pBI-11 DNA generates the greatest HPV antigen-specific CD8 + T cell immune responses and therapeutic antitumor effects compared to the other two methods. Monitoring of mouse behaviors and body weight, and necropsy indicated that electroporation-mediated delivery of clinical grade pBI-11 DNA vaccine was well-tolerated and presented no evident local or systemic toxicity.

Conclusions: These findings provide rationale for clinical testing of pBI-11 DNA vaccine delivered by electroporation for the control of HPV16/18-associated infections and/or cancers.

Background: Aberrant interplay between epigenetic reprogramming and metabolic rewiring events contributes to bladder cancer progression and metastasis. How the deacetylase Sirtuin-6 (SIRT6) regulates glycolysis and lactate secretion in bladder cancer remains poorly defined. We thus aimed to study the biological functions of SIRT6 in bladder cancer.

Methods: Bioinformatic analysis was used to study the prognostic significance of SIRT6/UHRF1 in BLCA. Both in vitro and in vivo assays were used to determine the roles of SIRT6/UHRF1 in BLCA. Deacetylation and ubiquitin assays were performed to uncover the regulations of SIRT6-UHRF1. Measurement of extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) was used to assess glycolytic abilities.

Results: Here, we show that protein deacetylase SIRT6 was down-regulated in BLCA, and predicts poor overall survival. SIRT6 deficiency notably enhances BLCA cell proliferation, self-renewal, and migration capacities in vitro and in vivo. Mechanistically, SIRT6 interacts with, deacetylates, and promotes UHRF1 degradation mediated by β-TrCP1. Thus, SIRT6 deficiency leads to stabilized UHRF1 and depends on UHRF1 to accelerate BLCA malignant progression. Furthermore, UHRF1 significantly increased aerobic glycolysis via activating MCT4/HK2 expressions. Down-regulated SIRT6 thus depended on UHRF1 to promote glycolysis and lactate secretion in BLCA. Targeting UHRF1 or MCT4 notably impaired the extracellular lactate accumulations in BLCA. Significantly, a specific small-molecule inhibitor (NSC232003) targeting UHRF1 substantially inhibited SIRT6-deficient BLCA progression.

Conclusion: Together, our study uncovered an epigenetic mechanism of the SIRT6/UHRF1 axis in driving BLCA glycolysis and lactate secretion, creating a novel vulnerability for BLCA treatment.

Background: c-Jun is a key regulator of gene expression. Through the formation of homo- or heterodimers, c-JUN binds to DNA and regulates gene transcription. While c-Jun plays a crucial role in embryonic development, its impact on nervous system development in higher mammals, especially for some deep structures, for example, thalamus in diencephalon, remains unclear.

Methods: To investigate the influence of c-JUN on early nervous system development, c-Jun knockout (KO) mice and c-JUN KO H1 embryonic stem cells (ESCs)-derived neural progenitor cells (NPCs), cerebral organoids (COs), and thalamus organoids (ThOs) models were used. We detected the dysplasia via histological examination and immunofluorescence staining, omics analysis, and loss/gain of function analysis.

Results: At embryonic day 14.5, c-Jun knockout (KO) mice exhibited sparseness of fibers in the brain ventricular parenchyma and malformation of the thalamus in the diencephalon. The absence of c-JUN accelerated the induction of NPCs but impaired the extension of fibers in human neuronal cultures. COs lacking c-JUN displayed a robust PAX6⁺/NESTIN⁺ exterior layer but lacked a fibers-connected core. Moreover, the subcortex-like areas exhibited defective thalamus characteristics with transcription factor 7 like 2-positive cells. Notably, in guided ThOs, c-JUN KO led to inadequate thalamus patterning with sparse internal nerve fibers. Chromatin accessibility analysis confirmed a less accessible chromatin state in genes related to the thalamus. Overexpression of c-JUN rescued these defects. RNA-seq identified 18 significantly down-regulated genes including RSPO2, WNT8B, MXRA5, HSPG2 and PLAGL1 while 24 genes including MSX1, CYP1B1, LMX1B, NQO1 and COL2A1 were significantly up-regulated.

Conclusion: Our findings from in vivo and in vitro experiments indicate that c-JUN depletion impedes the extension of nerve fibers and renders the thalamus susceptible to dysplasia during early mouse embryonic development and human ThO patterning. Our work provides evidence for the first time that c-JUN is a key transcription regulator that play important roles in the thalamus/diencephalon development.

Background: The Niemann Pick C1 (NPC1) protein is an intracellular cholesterol transporter located in the late endosome/lysosome (LE/Ly) that is involved in the mobilization of endocytosed cholesterol. Loss-of-function mutations in the NPC1 gene lead to the accumulation of cholesterol and sphingolipids in LE/Ly, resulting in severe fatal NPC1 disease. Cellular alterations associated with NPC1 inactivation affect both the integrity of lipid rafts and the endocytic pathway. Because the angiotensin-converting enzyme 2 (ACE2) and type 2 serine transmembrane protease (TMPRSS2), interactors of the SARS-CoV-2 Spike protein also localize to lipid rafts, we sought to investigate the hypothesis that NPC1 inactivation would generate an intrinsically unfavorable barrier to SARS-CoV-2 entry.

Results: In this study, we show that inhibition of the cholesterol transporter activity of NPC1 in cells that express both ACE2 and TMPRSS2, considerably reduces SARS-CoV-2 infectivity, evaluated as early as 4 h post-infection. Mechanistically, treatment with NPC1 specific inhibitor U18666A relocalizes ACE2 from the plasma membrane to the autophagosomal/lysosomal compartment, thereby reducing SARS-CoV-2 entry into treated cells. Reduction of viral entry was observed for both fully infectious SARS-CoV-2 virus and with a pseudotyped VSV-Spike-GFP virus. For instance, U18666A-treated Caco-2 cells infected with the pseudotyped VSV-Spike-GFP showed a > threefold and > 40-fold reduction in virus titer when infectivity was measured at 4 h or 24 h post-infection, respectively. A similar effect was observed in CRISP/R-Cas9-edited Caco-2 cells, which were even more resistant to SARS-CoV-2 infection as indicated by a 97% reduction of viral titers.

Conclusion: Overall, this study provides compelling evidence that the inhibition of NPC1 cholesterol transporter activity generates a cellular environment that hinders SARS-CoV-2 entry. ACE2 depletion from the plasma membrane appears to play a major role as limiting factor for viral entry.

Background: macrophage-targeting therapy of ischemic disease has made progress in clinic trial. However, the role and underlying mechanism of pro-inflammatory or anti-inflammatory polarized macrophages in modulating ischemic diseases remain incompletely understood.

Results: here we examine the effect of pro-inflammatory (LPS) and anti-inflammatory (IL-4) macrophage on ischemic diseases in a mouse ischemic hindlimb and heart model, and identify that signal regulatory protein α (Sirpα) modulates macrophage polarization induced angiogenesis via promoting phagocytosis or activating HIF1α nucleus relocation in macrophages, respectively. More importantly, the therapeutic effect of polarized macrophages is controlled by Sirpα in a time-dependent manner. Downregulation of macrophage Sirpα at the early-stage or upregulation of macrophage Sirpα at the late-stage of ischemic disease enhances the therapeutic effect. In contrast, increasing Sirpα at the early-stage or decreasing it at the late-stage leads to failure of inducing ischemic disease resilience. Mechanistically, we find that signal transducer and activator of transcription 3 and 6 (Stat3 and Stat6) mediate downregulation (pro-inflammatory polarization) or upregulation (anti-inflammatory polarization) of Sirpα, respectively.

Conclusion: Our results reveal that dynamic regulation of macrophage by Sirpα plays a critical role in alleviating ischemic diseases.

The inherent heterogeneity of tumor cells impedes the development of targeted therapies for specific glioblastoma (GBM) subtypes. This study aims to investigate the mesenchymal subtype of GBM to uncover detailed characteristics, potential therapeutic strategies, and improve precision treatment for GBM patients. We integrated single-cell RNA sequencing (scRNA-seq), single-nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq), and bulk RNA sequencing datasets to identify core gene modules, candidate therapeutic drugs, and key transcription factors specific to mesenchymal subtype GBM tumor cells which we validated in vitro and human samples. Our analysis encompassed a heterogeneous single-cell landscape of 55,845 cells from tumor and adjacent normal tissues, focusing on the mesenchymal subtype's adverse prognosis and its association with hypoxia. We identified a core gene module composed of 38 genes and, through pharmacogenomic analysis, found that Trametinib and Dasatinib exhibit increased effectiveness against mesenchymal subtype GBM cells. Furthermore, by incorporating snATAC-seq data, we delineated a crucial regulatory network and pinpointed the key transcription factor CEBPG. Our research has highlighted the strong link between the mesenchymal-like (MES-like) properties of GBM and hypoxia, providing valuable insights into candidate drugs and pivotal targets for precision treatment of the mesenchymal subtype.

Background: Diabetic polyneuropathy (DPN) is a common diabetes complication with limited treatment options. We aimed to identify circulating plasma proteins as potential therapeutic targets for DPN using Mendelian Randomization (MR).

Methods: The protein quantitative trait loci (pQTLs) utilized in this study were derived from seven previously published genome-wide association studies (GWASs) on plasma proteomics. The DPN data were obtained from the IEU OpenGWAS project. This study employed two-sample MR using MR-Egger and inverse-variance weighted methods to evaluate the causal relationship between plasma proteins and DPN risk, with Cochran's Q test, and I² statistics, among other methods, used to validate the robustness of the results.

Results: Using cis-pQTLs as genetic instruments, we identified 62 proteins associated with DPN, with 33 increasing the risk and 29 decreasing the risk of DPN. Using cis-pQTLs + trans-pQTLs, we identified 116 proteins associated with DPN, with 44 increasing the risk and 72 decreasing the risk of DPN. Steiger directionality tests indicated that the causal relationships between circulating plasma proteins and DPN were consistent with expected directions.

Conclusion: This study identified 96 circulating plasma proteins with genetically determined levels that affect the risk of DPN, providing new potential targets for DPN drug development, particularly ITM2B, CREG1, CD14, and PLXNA4.

Background: L-DOPA has been considered the first-line therapy for treating Parkinson's disease (PD) via restoring striatal dopamine (DA) to normalize the activity of local spiny projection neurons (SPNs) in the direct (dSPNs) pathway and the indirect (iSPNs) pathway. While the changes in striatal acetylcholine (ACh) induced by increasing DA have been extensively discussed, their validity remains controversial. Inhibition of striatal cholinergic signaling attenuates PD motor deficits. Interestingly, enhancing striatal ACh triggers local DA release, suggesting the pro-kinetic effects of ACh in movement control. Here, we investigated the in-vivo dynamics of ACh in the dorsolateral striatum (DLS) of the 6-OHDA-lesioned mouse model after L-DOPA administration, as well as its underlying mechanism, and to explore its modulatory role and mechanism in parkinsonian symptoms.

Results: Using in vivo fiber photometry recordings with genetically encoded fluorescent DA or ACh indicator, we found L-DOPA selectively decreased DLS ACh levels in parkinsonian conditions. DA inhibited ACh release via dopamine D2 receptors and dSPNs-mediated activation of type-A γ-aminobutyric acid receptors on cholinergic interneurons. Restoring DLS ACh levels during L-DOPA treatment induced additional DA release by activating nicotinic acetylcholine receptors, thereby promoting the activity of dSPNs and iSPNs. Enhancing DLS ACh facilitated L-DOPA-induced turning behavior but not dyskinesia in parkinsonian mice.

Conclusions: Our results demonstrated that enhancing striatal ACh facilitated the effect of L-DOPA by modulating DA tone. It may challenge the classical hypothesis of a purely competitive interaction between dopaminergic and cholinergic neuromodulation in improving PD motor deficits. Modulating ACh levels within the dopaminergic system may improve striatal DA availability in PD patients.