S-nitrosylation-triggered unfolded protein response maintains hematopoietic progenitors in Drosophila.

The Drosophila lymph gland houses blood progenitors that give rise to myeloid-like blood cells. Initially, blood progenitors proliferate, but later, they become quiescent to maintain multipotency before differentiation. Despite the identification of various factors involved in multipotency maintenance, the cellular mechanism controlling blood progenitor quiescence remains elusive. Here, we identify the expression of nitric oxide synthase in blood progenitors, generating nitric oxide for post-translational S-nitrosylation of protein cysteine residues. S-nitrosylation activates the Ire1-Xbp1-mediated unfolded protein response, leading to G2 cell-cycle arrest. Specifically, we identify the epidermal growth factor receptor as a target of S-nitrosylation, resulting in its retention within the endoplasmic reticulum and blockade of its receptor function. Overall, our findings highlight developmentally programmed S-nitrosylation as a critical mechanism that induces protein quality control in blood progenitors, maintaining their undifferentiated state by inhibiting cell-cycle progression and rendering them unresponsive to paracrine factors.

CYR61 confers chemoresistance by upregulating survivin expression in triple-negative breast cancer.

Cysteine-rich angiogenic inducer 61 (CYR61) is a protein from the CCN family of matricellular proteins that play diverse regulatory roles in the extracellular matrix. CYR61 is involved in cell adhesion, migration, proliferation, differentiation, apoptosis, and senescence. Here, we show that CYR61 induces chemoresistance in triple-negative breast cancer (TNBC). We observed that CYR61 is overexpressed in TNBC patients, and CYR61 expression correlates negatively with the survival of patients who receive chemotherapy. CYR61 knockdown reduced cell migration, sphere formation, and the cancer stem cell (CSC) population and increased the chemosensitivity of TNBC cells. Mechanistically, CYR61 activated Wnt/ß-catenin signaling and increased survivin expression, which are associated with chemoresistance, the epithelial-mesenchymal transition, and CSC-like phenotypes. Altogether, our study demonstrates a novel function of CYR61 in chemotherapy resistance in breast cancer.

Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization.

BACKGROUND: Solid tumors promote tumor malignancy through interaction with the tumor microenvironment, resulting in difficulties in tumor treatment. Therefore, it is necessary to understand the communication between cells in the tumor and the surrounding microenvironment. Our previous study revealed the cancer malignancy mechanism of Bcl-w overexpressed in solid tumors, but no study was conducted on its relationship with immune cells in the tumor microenvironment. In this study, we sought to discover key factors in exosomes secreted from tumors overexpressing Bcl-w and analyze the interaction with the surrounding tumor microenvironment to identify the causes of tumor malignancy. METHODS: To analyze factors affecting the tumor microenvironment, a miRNA array was performed using exosomes derived from cancer cells overexpressing Bcl-w. The discovered miRNA, miR-6794-5p, was overexpressed and the tumorigenicity mechanism was confirmed using qRT-PCR, Western blot, invasion, wound healing, and sphere formation ability analysis. In addition, luciferase activity and Ago2-RNA immunoprecipitation assays were used to study the mechanism between miR-6794-5p and its target gene SOCS1. To confirm the interaction between macrophages and tumor-derived miR-6794-5p, co-culture was performed using conditioned media. Additionally, immunohistochemical (IHC) staining and flow cytometry were performed to analyze macrophages in the tumor tissues of experimental animals. RESULTS: MiR-6794-5p, which is highly expressed in exosomes secreted from Bcl-w-overexpressing cells, was selected, and it was shown that the overexpression of miR-6794-5p increased migratory ability, invasiveness, and stemness maintenance by suppressing the expression of the tumor suppressor SOCS1. Additionally, tumor-derived miR-6794-5p was delivered to THP-1-derived macrophages and induced M2 polarization by activating the JAK1/STAT3 pathway. Moreover, IL-10 secreted from M2 macrophages increased tumorigenicity by creating an immunosuppressive environment. The in vitro results were reconfirmed by confirming an increase in M2 macrophages and a decrease in M1 macrophages and CD8+ T cells when overexpressing miR-6794-5p in an animal model. CONCLUSIONS: In this study, we identified changes in the tumor microenvironment caused by miR-6794-5p. Our study indicates that tumor-derived miR-6794-5p promotes tumor aggressiveness by inducing an immunosuppressive environment through interaction with macrophage.

Motor Interaction Control Based on Muscle Force Model and Depth Reinforcement Strategy.

The current motion interaction model has the problems of insufficient motion fidelity and lack of self-adaptation to complex environments. To address this problem, this study proposed to construct a human motion control model based on the muscle force model and stage particle swarm, and based on this, this study utilized the deep deterministic gradient strategy algorithm to construct a motion interaction control model based on the muscle force model and the deep reinforcement strategy. Empirical analysis of the human motion control model proposed in this study revealed that the joint trajectory correlation and muscle activity correlation of the model were higher than those of other comparative models, and its joint trajectory correlation was up to 0.90, and its muscle activity correlation was up to 0.84. In addition, this study validated the effectiveness of the motion interaction control model using the depth reinforcement strategy and found that in the mixed-obstacle environment, the model's desired results were obtained by training 1.1 × 103 times, and the walking distance was 423 m, which was better than other models. In summary, the proposed motor interaction control model using the muscle force model and deep reinforcement strategy has higher motion fidelity and can realize autonomous decision making and adaptive control in the face of complex environments. It can provide a theoretical reference for improving the effect of motion control and realizing intelligent motion interaction.

AptaTrans: a deep neural network for predicting aptamer-protein interaction using pretrained encoders.

BACKGROUND: Aptamers, which are biomaterials comprised of single-stranded DNA/RNA that form tertiary structures, have significant potential as next-generation materials, particularly for drug discovery. The systematic evolution of ligands by exponential enrichment (SELEX) method is a critical in vitro technique employed to identify aptamers that bind specifically to target proteins. While advanced SELEX-based methods such as Cell- and HT-SELEX are available, they often encounter issues such as extended time consumption and suboptimal accuracy. Several In silico aptamer discovery methods have been proposed to address these challenges. These methods are specifically designed to predict aptamer-protein interaction (API) using benchmark datasets. However, these methods often fail to consider the physicochemical interactions between aptamers and proteins within tertiary structures. RESULTS: In this study, we propose AptaTrans, a pipeline for predicting API using deep learning techniques. AptaTrans uses transformer-based encoders to handle aptamer and protein sequences at the monomer level. Furthermore, pretrained encoders are utilized for the structural representation. After validation with a benchmark dataset, AptaTrans has been integrated into a comprehensive toolset. This pipeline synergistically combines with Apta-MCTS, a generative algorithm for recommending aptamer candidates. CONCLUSION: The results show that AptaTrans outperforms existing models for predicting API, and the efficacy of the AptaTrans pipeline has been confirmed through various experimental tools. We expect AptaTrans will enhance the cost-effectiveness and efficiency of SELEX in drug discovery. The source code and benchmark dataset for AptaTrans are available at https://github.com/pnumlb/AptaTrans .

CCN3/NOV promotes metastasis and tumor progression via GPNMB-induced EGFR activation in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. TNBC patients typically exhibit unfavorable outcomes due to its rapid growth and metastatic potential. Here, we found overexpression of CCN3 in TNBC patients. We identified that CCN3 knockdown diminished cancer stem cell formation, metastasis, and tumor growth in vitro and in vivo. Mechanistically, ablation of CCN3 reduced activity of the EGFR/MAPK pathway. Transcriptome profiling revealed that CCN3 induces glycoprotein nonmetastatic melanoma protein B (GPNMB) expression, which in turn activates the EGFR pathway. An interrogation of the TCGA dataset further supported the transcriptional regulation of GPNMB by CCN3. Finally, we showed that CCN3 activates Wnt signaling through a ligand-dependent or -independent mechanism, which increases microphthalmia-associated transcription factor (MITF) protein, a transcription factor inducing GPNMB expression. Together, our findings demonstrate the oncogenic role of CCN3 in TNBC, and we propose CCN3 as a putative therapeutic target for TNBC.

MHC II immunogenicity shapes the neoepitope landscape in human tumors.

Despite advances in predicting physical peptide-major histocompatibility complex I (pMHC I) binding, it remains challenging to identify functionally immunogenic neoepitopes, especially for MHC II. By using the results of >36,000 immunogenicity assay, we developed a method to identify pMHC whose structural alignment facilitates T cell reaction. Our method predicted neoepitopes for MHC II and MHC I that were responsive to checkpoint blockade when applied to >1,200 samples of various tumor types. To investigate selection by spontaneous immunity at the single epitope level, we analyzed the frequency spectrum of >25 million mutations in >9,000 treatment-naive tumors with >100 immune phenotypes. MHC II immunogenicity specifically lowered variant frequencies in tumors under high immune pressure, particularly with high TCR clonality and MHC II expression. A similar trend was shown for MHC I neoepitopes, but only in particular tissue types. In summary, we report immune selection imposed by MHC II-restricted natural or therapeutic T cell reactivity.

Brain type of creatine kinase induces doxorubicin resistance via TGF-ß signaling in MDA-MB-231 breast cancer cells.

Brain type of creatine kinase (CKB) regulates energy homeostasis by reversibly transferring phosphate groups between phosphocreatine and ATP at sites of high energy demand. Several types of cancer cells exhibit upregulated CKB expression, but the function of CKB in cancer cells remains unclear. In this study, we investigated the function of CKB in breast cancer by overexpressing CKB in MDA-MB-231 cells. The overexpression of CKB did not affect cell growth rate, cell cycle distribution, ATP level or key mediators of aerobic glycolysis and lactate dehydrogenase isoform levels. Meanwhile, CKB overexpression did increase resistance to doxorubicin. TGF-ß-induced Smad phosphorylation and Smad-dependent transcriptional activity were significantly up-regulated by CKB expression without changes in inhibitory Smad protein levels. Moreover, treatment with TGF-ß considerably enhanced cell viability during doxorubicin treatment and decreased doxorubicin-induced apoptosis in CKB-expressing MDA-MB-231 cells compared to control cells. These results suggest that CKB attenuates doxorubicin-induced apoptosis and potentiates resistance to doxorubicin by enhancing TGF-ß signaling in MDA-MB-231 cells.

Glioblastoma-educated mesenchymal stem-like cells promote glioblastoma infiltration via extracellular matrix remodelling in the tumour microenvironment.

BACKGROUND: The biological function of mesenchymal stem-like cells (MSLCs), a type of stromal cells, in the regulation of the tumour microenvironment is unclear. Here, we investigated the molecular mechanisms underlying extracellular matrix (ECM) remodelling and crosstalk between MSLCs and glioblastomas (GBMs) in tumour progression. METHODS: In vitro and in vivo co-culture systems were used to analyze ECM remodelling and GBM infiltration. In addition, clinical databases, samples from patients with GBM and a xenografted mouse model of GBM were used. RESULTS: Previous studies have shown that the survival of patients with GBM from whom MSLCs could be isolated is substantially shorter than that of patients from whom MSLCs could not be isolated. Therefore, we determined the correlation between changes in ECM-related gene expression in MSLC-isolatable patients with that in MSLC non-isolatable patients using gene set enrichment analysis (GSEA). We found that lysyl oxidase (LOX) and COL1A1 expressions increased in MSLCs via GBM-derived clusters of differentiation 40 ligand (CD40L). Mechanistically, MSLCs are reprogrammed by the CD40L/CD40/NFκB2 signalling axis to build a tumour infiltrative microenvironment involving collagen crosslinking. Importantly, blocking of CD40L by a neutralizing antibody-suppressed LOX expression and ECM remodelling, decreasing GBM infiltration in mouse xenograft models. Clinically, high expression of CD40L, clusters of differentiation 40 (CD40) and LOX correlated with poor survival in patients with glioma. This indicated that GBM-educated MSLCs promote GBM infiltration via ECM remodelling in the tumour microenvironment. CONCLUSION: Our findings provide mechanistic insights into the pro-infiltrative tumour microenvironment produced by GBM-educated MSLCs and highlight a potential therapeutic target that can be used for suppressing GBM infiltration.

YAP, CTGF and Cyr61 are overexpressed in tamoxifen-resistant breast cancer and induce transcriptional repression of ERα.

About 70% of breast cancers overexpress estrogen receptor α (ERα, encoded by ESR1). Tamoxifen, a competitive inhibitor of estrogen that binds to ER, has been widely used as a treatment for ER-positive breast cancer. However, 20-30% of breast cancer is resistant to tamoxifen treatment. The mechanisms underlying tamoxifen resistance remain elusive. We found that Yes-associated protein (YAP; also known as YAP1), connective tissue growth factor (CTGF; also known as CCN2) and cysteine-rich angiogenic inducer 61 (Cyr61; also known as CCN1) are overexpressed, while ERα is downregulated in tamoxifen-resistant breast cancer. Inhibition of YAP, CTGF and Cyr61 restored ERα expression and increased sensitivity to tamoxifen. Overexpression of YAP, CTGF, and Cyr61 led to downregulation of ERα and conferred resistance to tamoxifen in ER-positive breast cancer cells. Mechanistically, CTGF and Cyr61 downregulated ERα expression at the transcriptional level by directly binding to the regulatory regions of the ERα-encoding gene, leading to increased tamoxifen resistance. Also, CTGF induced Glut3 (also known as SLC2A3) expression, leading to increased glycolysis, which enhanced cell proliferation and migration in tamoxifen-resistant cells. Together, these results demonstrate a novel role of YAP, CTGF and Cyr61 in tamoxifen resistance and provide a molecular basis for their function in tamoxifen-resistant breast cancer.

HER2 Signaling in Breast Cancer.

HER2 gene amplification occurs in many breast cancer patients and is associated with poor clinical prognosis. Trastuzumab is a therapeutic monoclonal antibody binding to HER2 and inhibits growth of HER2-positive breast cancer cells and used as a principal treatment for HER2-positive breast cancer. Unfortunately, some HER2-positive breast cancers eventually relapse after trastuzumab treatment. To investigate the molecular mechanism of trastuzumab resistance, we generated trastuzumab-resistant cells using a mouse model and found ECM1 protein is increased in trastuzumab-resistant cells. ECM1 was shown to increase EGFR signaling via upregulated matrix metalloproteinase 9/galectin-3/mucin pathway. To further find the novel mediators of HER2-driven signaling pathways in breast cancer, we investigated the upregulated proteins in HER2-overexpressing breast cancer cells using a proteomics approach and found that KRT19 is strongly upregulated in HER2-positive breast cancer cells and it activates HER2 signaling by binding to HER2 and stabilizes the receptor on the cell membrane. Moreover, we found that treatment of KRT19 antibody resulted in reduced cell viability of trastuzumab-resistant HER2-positive breast cancer cells as well as trastuzumab-sensitive cancer cells both in vitro and in vivo.

CTGF regulates cell proliferation, migration, and glucose metabolism through activation of FAK signaling in triple-negative breast cancer.

Connective tissue growth factor (CTGF), also known as CCN2, is a member of the CCN protein family of secreted proteins with roles in diverse biological processes. CTGF regulates biological functions such as cell proliferation, migration, adhesion, wound healing, and angiogenesis. In this study, we demonstrate a mechanistic link between CTGF and enhanced aerobic glycolysis in triple-negative breast cancer (TNBC). We found that CTGF is overexpressed in TNBC and high CTGF expression is correlated with a poor prognosis. Also, CTGF was required for in vivo tumorigenesis and in vitro proliferation, migration, invasion, and adhesion of TNBC cells. Our results indicate that extracellular CTGF binds directly to integrin αvß3, activating the FAK/Src/NF-κB p65 signaling axis, which results in transcriptional upregulation of Glut3. Neutralization of CTGF decreased cell proliferation, migration, and invasion through downregulation of Glut3-mediated glycolytic phenotypes. Overall, our work suggests a novel function for CTGF as a modulator of cancer metabolism, indicating that CTGF is a potential therapeutic target in TNBC.

Mass Cytometry and Single-Cell RNA-seq Profiling of the Heterogeneity in Human Peripheral Blood Mononuclear Cells Interacting with Silver Nanoparticles.

Understanding the interactions between nanoparticles (NPs) and human immune cells is necessary for justifying their utilization in consumer products and biomedical applications. However, conventional assays may be insufficient in describing the complexity and heterogeneity of cell-NP interactions. Herein, mass cytometry and single-cell RNA-sequencing (scRNA-seq) are complementarily used to investigate the heterogeneous interactions between silver nanoparticles (AgNPs) and primary immune cells. Mass cytometry reveals the heterogeneous biodistribution of the positively charged polyethylenimine-coated AgNPs in various cell types and finds that monocytes and B cells have higher association with the AgNPs than other populations. scRNA-seq data of these two cell types demonstrate that each type has distinct responses to AgNP treatment: NRF2-mediated oxidative stress is confined to B cells, whereas monocytes show Fcγ-mediated phagocytosis. Besides the between-population heterogeneity, analysis of single-cell dose-response relationships further reveals within-population diversity for the B cells and naïve CD4+ T cells. Distinct subsets having different levels of cellular responses with respect to their cellular AgNP doses are found. This study demonstrates that the complementary use of mass cytometry and scRNA-seq is helpful for gaining in-depth knowledge on the heterogeneous interactions between immune cells and NPs and can be incorporated into future toxicity assessments of nanomaterials.

Silencing of CD133 inhibits GLUT1-mediated glucose transport through downregulation of the HER3/Akt/mTOR pathway in colon cancer.

Cluster of differentiation 133 (CD133) is a transmembrane glycoprotein that has been reported as a marker of cancer stem cells or cancer-initiating cells in various cancers. However, its contribution to tumorigenesis and differentiation remains to be elucidated. To determine the role of CD133 in colon cancer, we silenced CD133 in human colon cancer cells. Silencing of CD133 results in decreased cell proliferation, survival, migration, invasion, and glucose transport. These effects are mediated by downregulation of the human epidermal growth factor receptor 3 (HER3)/Akt/mTOR signaling pathway, culminating in reduced expression of the glucose transporter GLUT1. We also confirm that the cellular phenotypes of CD133-silenced cells are mediated by GLUT1 downregulation. We conclude that CD133 is a potential tumor initiator that positively regulates GLUT1 expression through modulation of HER3/Akt/mTOR signaling.

Toxicological assessment of phthalates and their alternatives using human keratinocytes.

Phthalates are mainly used as binders and plasticizers in various industrial products including detergents, surfactants, waxes, paints, pharmaceuticals, food products, and cosmetics. However, they have been reported to be endocrine disruptors, which are chemicals that can mimic or disturb endocrines, causing interference to the endocrine system. Recently, there have been numerous reports showing that phthalates have negative health impacts such as asthma, breast cancer, obesity, type II diabetes, and male infertility. Due to these effects, there is an urgent need for phthalate alternatives. In this study, the potential cytotoxicity of phthalates and their substitutes were screened in HaCaT cells, a human keratinocyte cell line, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) thiazolyl blue assay, immunocytochemistry, flow cytometric analysis, and western blotting. We confirmed that common phthalates such as butyl benzyl phthalate (BBP), di-n-butyl phthalate (DBP), and di-2-ethylhexyl phthalate (DEHP) have genotoxic effects, leading to cell death. Among the known phthalate substitutes, tributyl O-acetylcitrate (ATBC), triethyl 2-acetylcitrate (ATEC), and trihexyl O-acetylcitrate (ATHC) were tested for cytotoxicity. As a result, ATEC showed similar levels of cytotoxicity with the phthalates whereas ATBC and ATHC did not show significant cytotoxicity even in high doses (5 mg/ml).

Titanium dioxide nanoparticles induce apoptosis by interfering with EGFR signaling in human breast cancer cells.

Titanium dioxide nanoparticles, due to their smaller size and increased surface area comparted to the bulk form, are known to be bioreactive and have unexpected toxicological outcomes. Previous studies have shown that nanoscale titanium dioxide induces reactive oxygen species (ROS)-mediated cytotoxicity and genotoxicity. Although many reports have discussed the ROS-mediated cytotoxic effects of titanium dioxide nanoparticles (TiO2-NPs), their effects on the receptor-ligand association are unknown. In this study, the possibility that TiO2-NPs can interfere with the receptor-ligand binding was assessed by monitoring alterations in the phosphorylation status of proteins downstream of the epidermal growth factor receptor (EGFR) signaling cascade. TiO2-NPs blocked ligand-induced EGFR autophosphorylation, leading to the deactivation of EGFR downstream effectors such as Akt and extracellular signal-regulated kinase signaling, inducing cell death.

Correction to: ECM1 regulates cell proliferation and trastuzumab resistance through activation of EGF-signaling.

After the publication of this work [1] errors were found in Figs. 1a and 5d.

Role of the CCN protein family in cancer.

The CCN protein family is composed of six matricellular proteins, which serve regulatory roles rather than structural roles in the extracellular matrix. First identified as secreted proteins which are induced by oncogenes, the acronym CCN came from the names of the first three members: CYR61, CTGF, and NOV. All six members of the CCN family consist of four cysteine-rich modular domains. CCN proteins are known to regulate cell adhesion, proliferation, differentiation, and apoptosis. In addition, CCN proteins are associated with cardiovascular and skeletal development, injury repair, inflammation, and cancer. They function either through binding to integrin receptors or by regulating the expression and activity of growth factors and cytokines. Given their diverse roles related to the pathology of certain diseases such as fibrosis, arthritis, atherosclerosis, diabetic nephropathy, retinopathy, and cancer, there are many emerging studies targeting CCN protein signaling pathways in attempts to elucidate their potentials as therapeutic targets. [BMB Reports 2018; 51(10): 486-493].

Cytotoxicity measurement of Bisphenol A (BPA) and its substitutes using human keratinocytes.

Bisphenol-A (BPA) was first synthesized in the 1890s and has been used in many plastic products. However, BPA is known to act as an endocrine disruptor and has been found to be toxic to human health. Many alternative substances have been developed to replace BPA, but it is still widely used worldwide. In this study, we identified the potential cytotoxicity of BPA by evaluating toxicity using human keratinocytes. Also, we evaluated cytotoxicity of BPA substitutes to determine their suitability as an alternative to BPA. The proliferation assay using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and western blot analysis showed that BPA significantly affect cell viability, induction of apoptotic fraction and increased activation of DNA-damage marker protein. In addition, through the same experiments, the substitutes of BPA were shown to be significantly less toxic than BPA, and the least toxicity was observed with 1,4-cyclohexanedimethanol (CHDM) and terephthalic acid (TPA). In conclusion, this study suggests that cytotoxicity of BPA induces apoptosis of human keratinocytes, and that CHDM and TPA are the most suitable substitutes for BPA.

Egr-1 is required for neu/HER2-induced mammary tumors.

Egr-1 is known to function mainly as a tumor suppressor through direct regulation of multiple tumor suppressor genes. To determine the role of Egr-1 in breast tumors in vivo, we used mouse models of breast cancer induced by HER2/neu. We compared neu-overexpressing Egr-1 knockout mice (neu/Egr-1 KO) to neu-overexpressing Egr-1 wild type or heterozygote mice (neu/Egr-1 WT or neu/Egr-1 het) with regard to onset of tumor appearance and number of tumors per mouse. In addition, to examine the role of Egr-1 in vitro, we established neu/Egr-1 WT and KO tumor cell lines derived from breast tumors developed in each mouse. Egr-1 deletion delayed tumor development in vivo and decreased the rate of cell growth in vitro. These results suggest that Egr-1 plays an oncogenic role in HER2/neu-driven mammary tumorigenesis.