Piperlongumine-inhibited TRIM14 signaling sensitizes glioblastoma cells to temozolomide treatment.

AIMS: Glioblastoma multiforme (GBM) is the most aggressive and mortal primary glioma in adults. Temozolomide (TMZ) is a first-line clinical chemotherapeutic drug. However, TMZ resistance causes treatment failure in patients. Thus, exploring effective adjuvant drugs for GBM is crucial. Piperlongumine (PL), a bioactive alkaloid isolated from long pepper, possesses promising anticancer abilities. However, PL-mediated cytotoxic mechanisms in GBM are still unclear. We attempted to identify PL-regulated networks in suppressing GBM malignancy. MAIN METHODS AND KEY FINDINGS: PL treatment significantly induced more apoptotic death in several GBM cell lines than in normal astrocytes. Decreased cell invasion, colony generation, and sphere formation, and enhanced TMZ cytotoxicity were found in PL-treated cells. Through RNA sequencing, PL-mediated transcriptomic profiles were established. By intersecting PL-downregulated genes, higher expressing genes in The Cancer Genome Atlas (TCGA) tumor tissues, and risk genes in three different GBM databases, tripartite motif-containing 14 (TRIM14) was selected. Higher TRIM14 expression was correlated with poor patient survival, and it existed in tumor samples, in mesenchymal type of GBM patients, and in GBM cells. PL significantly reduced TRIM14 expression through activating the p38/MAPK pathway. Overexpression or knockdown of TRIM14 influenced cell growth, PL-inhibited cell viability, invasion, colony generation, and sphere formation. Finally, using a gene set enrichment analysis, genes positively correlated with TRIM14 levels were enriched in epithelial-to-mesenchymal transition signaling. TRIM14 overexpression attenuated PL-regulated mesenchymal transition signaling. SIGNIFICANCE: PL inhibited TRIM14 signaling through activating the p38/MAPK pathway to inhibit GBM malignancy. Our findings may provide better insights and directions for future GBM therapies.

miR-4286 is Involved in Connections Between IGF-1 and TGF-ß Signaling for the Mesenchymal Transition and Invasion by Glioblastomas.

The insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-ß signal pathways are both recognized as important in regulating cancer prognosis, such as the epithelial-to-mesenchymal transition (EMT) and cell invasion. However, cross-talk between these two signal pathways in glioblastoma multiforme (GBM) is still unclear. In the present study, by analyzing data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GSE) 4412, GBM patients with higher IGF-1 levels exhibited poorer survival. Genes positively correlated with IGF-1 were enriched in EMT and TGF-ß signal pathways. IGF-1 treatment enhanced mesenchymal marker expressions and GBM cell invasion. A significant positive correlation was observed for IGF-1 with TGF-ß1 (TGFB1) or TGF-ß receptor 2 (TGFBR2), both of which participate in TGF-ß signaling and are risk genes in the GBM process. IGF-1 stimulation promoted both TGFB1 and TGFBR2 expressions. LY2157299, a TGF-ß signaling inhibitor, attenuated IGF-1-enhanced GBM cell invasion and mesenchymal transition. By analyzing IGF-1-regulated microRNA (miR) profiles, miR-4286 was found to be significantly downregulated in IGF-1-treated cells and could be targeted to both TGFB1 and TGFBR2. Overexpression of miR-4286 significantly attenuated expressions of the IGF-1-mediated mesenchymal markers, TGFB1 and TGFBR2. Using kinase inhibitors, only U0126 treatment showed an inhibitory effect on IGF-1-reduced miR-4286 and IGF-1-induced TGFB1/TGFBR2 expressions, suggesting that MEK/ERK signaling is involved in the IGF-1/miR-4286/TGF-ß signaling axis. Finally, our results suggested that miR-4286 might act as a tumor suppressive microRNA in inhibiting IGF-1-enhanced GBM cell invasion. In conclusion, IGF-1 is connected to TGF-ß signaling in regulating the mesenchymal transition and cell invasion of GBM through inhibition of miR-4286. Our findings provide new directions and mechanisms for exploring GBM progression.

Hypoxia-inducible lncRNA MIR210HG interacting with OCT1 is involved in glioblastoma multiforme malignancy.

An insufficient oxygen supply within the intratumoral environment, also known as hypoxia, induces glioblastoma multiforme (GBM) invasion, stemness, and temozolomide (TMZ) drug resistance. Long noncoding (lnc)RNAs have been reported to be involved in hypoxia and GBM progression. However, their roles in hypoxic GBM malignancy are still unclear. We investigated the mechanisms of hypoxia-mediated lncRNAs in regulating GBM processes. Using The Cancer Genome Atlas (TCGA) and data mining, hypoxia-correlated lncRNAs were identified. A hypoxia-upregulated lncRNA, MIR210HG, locating in nuclear regions, predicted poor prognoses of patients and modulated hypoxia-promoted glioma stemness, TMZ resistance, and invasion. Depletion of hypoxic MIR210HG suppressed GBM and patient-derived cell growth and increased TMZ sensitivity in vitro and vivo. Using RNA sequencing and gene set enrichment analysis (GSEA), MIR210HG-upregulated genes significantly belonged to the targets of octamer transcription factor 1 (OCT1) transcription factor. The direct interaction between OCT1 and MIR210HG was also validated. Two well-established worse prognostic factors of GBM, insulin-like growth factor-binding protein 2 (IGFBP2) and fibroblast growth factor receptor 1 (FGFR1), were identified as downstream targets of OCT1 through MIR210HG mediation in hypoxia. Consequently, the lncRNA MIR210HG is upregulated by hypoxia and interacts with OCT1 for modulating hypoxic GBM, leading to poor prognoses. These findings might provide a better understanding in functions of hypoxia/MIR210HG signaling for regulating GBM malignancy.

A regulatory loop among CD276, miR-29c-3p, and Myc exists in cancer cells against natural killer cell cytotoxicity.

AIMS: Immune checkpoints regulate immunity to prevent autoimmunity and protect the host from damage during pathogenic infection. They also participate in subverting immune surveillance and promote antitumor immunity in cancers. Although immunotherapy improves clinical outcomes, not all cancer patients experience expected responses after therapy. Hence, it would be meaningful to explore crucial immune checkpoints in cancers for future immunotherapies. METHODS AND KEY FINDINGS: By analyzing pan-cancer data in The Cancer Genome Atlas (TCGA), cluster of differentiation 276 (CD276), also known as B7H3, was found to be a risk gene in several cancers. A positive correlation existed between CD276 and natural killer (NK) cell infiltration. Overexpression of CD276 attenuated NK cell-mediated cell killing. Furthermore, CD276 levels showed a significant negative association with microRNA (miR)-29c-3p. Overexpression of miR-29c-3p rescued CD276-reduced NK cell cytotoxicity. According to gene set enrichment analyses, CD276-associated genes were found to be enriched in genes that targeted Myc. A negative correlation existed between miR-29 expression and Myc activity. CD276 enhanced Myc phosphorylation levels while suppressing miR-29c-3p expression. In contrast, miR-29c-3p inhibited CD276 expression, leading to reduced Myc activity. Myc suppressed miR-29c-3p expression while promoting CD276 upregulation. SIGNIFICANCE: These findings suggest that a negative regulatory loop among CD276, Myc, and miR-29c-3p influences cancer cells against NK cell cytotoxicity.

Cancer Essential Genes Stratified Lung Adenocarcinoma Patients with Distinct Survival Outcomes and Identified a Subgroup from the Terminal Respiratory Unit Type with Different Proliferative Signatures in Multiple Cohorts.

Background: Heterogeneous features of lung adenocarcinoma (LUAD) are used to stratify patients into terminal respiratory unit (TRU), proximal-proliferative (PP), and proximal-inflammatory (PI) subtypes. A more-accurate subtype classification would be helpful for future personalized medicine. However, these stratifications are based on genes with variant expression levels without considering their tumor-promoting roles. We attempted to identify cancer essential genes for LUAD stratification and their clinical and biological differences. Methods: Essential genes in LUAD were identified using genome-scale CRIPSR screening of RNA sequencing data from Project Achilles and The Cancer Genome Atlas (TCGA). Patients were stratified using consensus clustering. Survival outcomes, genomic alterations, signaling activities, and immune profiles within clusters were investigated using other independent cohorts. Findings: Thirty-six genes were identified as essential to LUAD, and there were used for stratification. Essential gene-classified clusters exhibited distinct survival rates and proliferation signatures across six cohorts. The cluster with the worst prognosis exhibited TP53 mutations, high E2F target activities, and high tumor mutation burdens, and harbored tumors vulnerable to topoisomerase I and poly(ADP ribose) polymerase inhibitors. TRU-type patients could be divided into clinically and molecularly different subgroups based on these essential genes. Conclusions: Our study showed that essential genes to LUAD not only defined patients with different survival rates, but also refined preexisting subtypes.

Glycolysis-associated lncRNAs identify a subgroup of cancer patients with poor prognoses and a high-infiltration immune microenvironment.

BACKGROUND: Long noncoding (lnc)RNAs and glycolysis are both recognized as key regulators of cancers. Some lncRNAs are also reportedly involved in regulating glycolysis metabolism. However, glycolysis-associated lncRNA signatures and their clinical relevance in cancers remain unclear. We investigated the roles of glycolysis-associated lncRNAs in cancers. METHODS: Glycolysis scores and glycolysis-associated lncRNA signatures were established using a single-sample gene set enrichment analysis (GSEA) of The Cancer Genome Atlas pan-cancer data. Consensus clustering assays and genomic classifiers were used to stratify patient subtypes and for validation. Fisher's exact test was performed to investigate genomic mutations and molecular subtypes. A differentially expressed gene analysis, with GSEA, transcription factor (TF) activity scoring, cellular distributions, and immune cell infiltration, was conducted to explore the functions of glycolysis-associated lncRNAs. RESULTS: Glycolysis-associated lncRNA signatures across 33 cancer types were generated and used to stratify patients into distinct clusters. Patients in cluster 3 had high glycolysis scores and poor survival, especially in bladder carcinoma, low-grade gliomas, mesotheliomas, pancreatic adenocarcinomas, and uveal melanomas. The clinical significance of lncRNA-defined groups was validated using external datasets and genomic classifiers. Gene mutations, molecular subtypes associated with poor prognoses, TFs, oncogenic signaling such as the epithelial-to-mesenchymal transition (EMT), and high immune cell infiltration demonstrated significant associations with cluster 3 patients. Furthermore, five lncRNAs, namely MIR4435-2HG, AC078846.1, AL157392.3, AP001273.1, and RAD51-AS1, exhibited significant correlations with glycolysis across the five cancers. Except MIR4435-2HG, the lncRNAs were distributed in nuclei. MIR4435-2HG was connected to glycolysis, EMT, and immune infiltrations in cancers. CONCLUSIONS: We identified a subgroup of cancer patients stratified by glycolysis-associated lncRNAs with poor prognoses, high immune infiltration, and EMT activation, thus providing new directions for cancer therapy.

Guanabenz Sensitizes Glioblastoma Cells to Sunitinib by Inhibiting GADD34-Mediated Autophagic Signaling.

Limited therapeutic efficacy of temozolomide (TMZ) against glioblastomas highlights the importance of exploring new drugs for clinical therapy. Sunitinib, a multitargeted receptor tyrosine kinase inhibitor, is currently being tested as therapy for glioblastomas. Unfortunately, sunitinib still has insufficient activity to cure glioblastomas. Our aim was to determine the molecular mechanisms counteracting sunitinib drug sensitivity and find potential adjuvant drugs for glioblastoma therapy. Through in vitro experiments, transcriptome screening by RNA sequencing, and in silico analyses, we found that sunitinib induced glioma apoptotic death, and downregulated genes were enriched in oncogenic genes of glioblastoma. Meanwhile, sunitinib-upregulated genes were highly associated with the protective autophagy process. Blockade of autophagy significantly enhanced sunitinib's cytotoxicity. Growth arrest and DNA damage-inducible protein (GADD) 34 was identified as a candidate involved in sunitinib-promoted autophagy through activating p38-mitogen-activated protein kinase (MAPK) signaling. Higher GADD34 levels predicted poor survival of glioblastoma patients and induced autophagy formation in desensitizing sunitinib cytotoxicity. Guanabenz, an alpha2-selective adrenergic agonist and GADD34 functional inhibitor, was identified to enhance the efficacy of sunitinib by targeting GADD34-induced protective autophagy in glioblastoma cells, TMZ-resistant cells, hypoxic cultured cells, sphere-forming cells, and colony formation abilities. A better combined treatment effect with sunitinib and guanabenz was also observed by using xenograft mice. Taken together, the sunitinib therapy combined with guanabenz in the inhibition of GADD34-enhanced protective autophagy may provide a new therapeutic strategy for glioblastoma.

Xanthohumol regulates miR-4749-5p-inhibited RFC2 signaling in enhancing temozolomide cytotoxicity to glioblastoma.

AIMS: Xanthohumol (XN), a natural prenylated flavonoid isolated from Humulus lupulus L. (hops), possess the therapeutic effects in glioblastoma multiforme (GBM), which is a grade IV aggressive glioma in adults. However, low bioavailability and extractive yield limit the clinical applications of XN. To comprehensively investigate XN-mediated gene networks in inducing cell death is helpful for drug development and cancer research. Therefore, we aim to identify the detailed molecular mechanisms of XN's effects on exhibiting cytotoxicity for GBM therapy. METHODS AND KEY FINDINGS: XN significantly induced GBM cell death and enhanced temozolomide (TMZ) cytotoxicity, a first-line therapeutic drug of GBM. XN-mediated transcriptome profiles and canonical pathways were identified. DNA repair signaling, a well-established mechanism against TMZ cytotoxicity, was significantly correlated with XN-downregulated genes. Replication factor C subunit 2 (RFC2), a DNA repair-related gene, was obviously downregulated in XN-treated cells. Higher RFC2 levels which occupied poor patient survival were also observed in high grade GBM patients and tumors. Inhibition of RFC2 reduced cell viability, induced cell apoptosis, and enhanced both XN and TMZ cytotoxicity. By intersecting array data, bioinformatic prediction, and in vitro experiments, microRNA (miR)-4749-5p, a XN-upregulated microRNA, was identified to target to RFC2 3'UTR and inhibited RFC2 expression. A negative correlation existed between miR-4749-5p and RFC2 in GBM patients. Overexpression of miR-4749-5p significantly promoted XN- and TMZ-mediated cytotoxicity, and reduced RFC2 levels. SIGNIFICANCE: Consequently, we suggest that miR-4749-5p targeting RFC2 signaling participates in XN-enhanced TMZ cytotoxicity of GBM. Our findings provide new potential therapeutic directions for future GBM therapy.

A Key Role of DNA Damage-Inducible Transcript 4 (DDIT4) Connects Autophagy and GLUT3-Mediated Stemness To Desensitize Temozolomide Efficacy in Glioblastomas.

DNA damage-inducible transcript 4 (DDIT4) is known to participate in various cancers, including glioblastoma multiforme (GBM). However, contradictory roles of DDIT4 exist in inducing cell death and possessing anti-apoptotic functions against cancer progression. Herein, we investigated DDIT4 signaling in GBM and temozolomide (TMZ) drug resistance. We identified that TMZ induced DDIT4 upregulation, leading to desensitization against TMZ cytotoxicity in GBM cells. Higher DDIT4 levels were found in glioma cells and mesenchymal-type GBM patients, and these higher levels were positively correlated with mesenchymal markers. Furthermore, patients with lower DDIT4 levels, especially O-6-methylguanine-DNA methyltransferase (MGMT)-methylated patients, exhibited better TMZ therapeutic efficacy. We determined that higher levels of 5 DDIT4-associated downstream genes, including SLC2A3 (also known as glucose transporter 3 (GLUT3)), can be used to predict a poor prognosis. Among these 5 genes, only GLUT3 was upregulated in both TMZ-treated and DDIT4-overexpressing cells. DDIT4-mediated GLUT3 expression was also identified, and its expression decreased TMZ's cytotoxicity. A significant correlation existed between DDIT4 and GLUT3. DDIT4 signaling was found to be involved in both glycolytic and autophagic pathways. However, GLUT3 only participated in the exhibition of DDIT4-mediated stemness, resulting from glycolytic regulation, but not in DDIT4-mediated autophagic signaling. Finally, we identified TMZ-upregulated activating transcription factor 4 (ATF4) as an upstream regulator of DDIT4-mediated GLUT3/stemness signaling and autophagy. Consequently, ATF4/DDIT4 signaling was connected to both autophagy and GLUT3-regulated stemness, which are involved in TMZ drug resistance and the poor prognoses of GBM patients. Targeting DDIT4/GLUT3 signaling might be a new direction for glioma therapy.

IGF-1-enhanced miR-513a-5p signaling desensitizes glioma cells to temozolomide by targeting the NEDD4L-inhibited Wnt/ß-catenin pathway.

Temozolomide (TMZ) is a first-line alkylating agent for glioblastoma multiforme (GBM). Clarifying the mechanisms inducing TMZ insensitivity may be helpful in improving its therapeutic effectiveness against GBM. Insulin-like growth factor (IGF)-1 signaling and micro (mi)RNAs are relevant in mediating GBM progression. However, their roles in desensitizing GBM cells to TMZ are still unclear. We aimed to identify IGF-1-mediated miRNA regulatory networks that elicit TMZ insensitivity for GBM. IGF-1 treatment attenuated TMZ cytotoxicity via WNT/ß-catenin signaling, but did not influence glioma cell growth. By miRNA array analyses, 93 upregulated and 148 downregulated miRNAs were identified in IGF-1-treated glioma cells. miR-513a-5p from the miR-513a-2 gene locus was upregulated by IGF-1-mediated phosphoinositide 3-kinase (PI3K) signaling. Its elevated levels were also observed in gliomas versus normal cells, in array data of The Cancer Genome Atlas (TCGA), and the GSE61710, GSE37366, and GSE41032 datasets. In addition, lower levels of neural precursor cell-expressed developmentally downregulated 4-like (NEDD4L), an E3 ubiquitin protein ligase that inhibits WNT signaling, were found in gliomas by analyzing cells, arrays, and RNA sequencing data of TCGA glioma patients. Furthermore, a negative correlation was identified between miR-513a-5p and NEDD4L in glioma. NEDD4L was also validated as a direct target gene of miR-513a-5p, and it was reduced by IGF-1 treatment. Overexpression of NEDD4L inhibited glioma cell viability and reversed IGF-1-repressed TMZ cytotoxicity. In contrast, miR-513a-5p significantly affected NEDD4L-inhibited WNT signaling and reduced TMZ cytotoxicity. These findings demonstrate a distinct role of IGF-1 signaling through miR-513a-5p-inhibited NEDD4L networks in influencing GBM's drug sensitivity to TMZ.

miR-140 targeting CTSB signaling suppresses the mesenchymal transition and enhances temozolomide cytotoxicity in glioblastoma multiforme.

Temozolomide (TMZ) is a first-line chemotherapeutic agent used against glioblastoma multiforme (GBM), but this disease exhibits recurrence and high lethality. Therefore, it is critical to explore biomarkers which involve in drug resistance and can be represented as different therapeutic effects after a diagnosis. We attempted to investigate the underlying variably expressed genes that contribute to the formation of resistance to TMZ. We analyzed gene and microRNA (miR) data from GBM patients in The Cancer Genome Atlas (TCGA) database to identify genetic factors associated with poor TMZ efficacy. By conducting a gene set enrichment analysis (GSEA), the epithelial-to-mesenchymal transition (EMT) was associated with poor TMZ responses. To identify roles of microRNAs in regulating TMZ resistance, a differential microRNA analysis was performed in TMZ-treated GBM patients. Downregulation of miR-140 was significantly correlated with poor survival. By integrating TCGA transcriptomic data and genomics of drug sensitivity in cancer (GDSC), cathepsin B (CTSB) was inversely associated with miR-140 expression and poor TMZ efficacy. By a pan-cancer analysis, both miR-140 and CTSB were found to be prognostic factors in other cancer types. We also identified that CTSB was a direct target gene of miR-140. Overexpression of miR-140 reduced CTSB levels, enhanced TMZ cytotoxicity, suppressed the mesenchymal transition, and influenced CTSB-regulated tumor sphere formation and stemness marker expression. In contrast, overexpression of CTSB decreased TMZ-induced glioma cell death, promoted the mesenchymal transition, and attenuated miR-140-increased TMZ cytotoxicity. These findings provide novel targets to increase the therapeutic efficacy of TMZ against GBM.

Gene landscape and correlation between B-cell infiltration and programmed death ligand 1 expression in lung adenocarcinoma patients from The Cancer Genome Atlas data set.

Tumor-infiltrating lymphocytes are related to positive clinical prognoses in numerous cancer types. Programmed death ligand 1 (PD-L1), a mediator of the PD-1 receptor, plays an inhibitory role in cancer immune responses. PD-L1 upregulation can impede infiltrating T-cell functions in lung adenocarcinoma (LUAD), a lung cancer subtype. However, associations between the expression of PD-L1 and infiltration of B cells (a major immunoregulatory cell) remain unknown. Therefore, we investigated the role of infiltrating B cells in LUAD progression and its correlation with PD-L1 expression. The Cancer Genome Atlas (TCGA) LUAD data set was used to explore associations among B-cell infiltration, PD-L1 expression, clinical outcome, and gene landscape. Gene set enrichment analysis was used to explore putative signaling pathways and candidate genes. The drug enrichment analysis was used to identify candidate genes and the related drugs. We found that high B-cell infiltration was correlated with better prognoses; however, PD-L1 may interfere with the survival advantage in patients with high B-cell infiltration. The gene landscape was characterized comprehensively, with distinct PD-L1 levels in cell populations with high B-cell infiltration. We obtained five upregulated signaling pathways from the gene landscape: apoptosis, tumor necrosis factor (TNF)-α signaling via nuclear factor (NF)-κB, apical surface, interferon-α response, and KRAS signaling. Moreover, four candidate genes and their related target drugs were also identified, namely interleukin-2ß receptor (IL2RB), IL-2γ receptor (IL2RG), Toll-like receptor 8 (TLR8), and TNF. These findings suggest that tumor-infiltrating B cells could act as a clinical factor in anti-PD-L1 immunotherapy for LUAD.

microRNA-199a/b-5p enhance imatinib efficacy via repressing WNT2 signaling-mediated protective autophagy in imatinib-resistant chronic myeloid leukemia cells.

Imatinib (IM) is a first-line therapeutic drug for chronic myeloid leukemia (CML), a hematological disease. Mutations in the BCR-ABL domain increase formation of IM resistance in CML. However, not all patients are BCR-ABL domain-mutant dependent. Investigating non-mutant mechanisms in the development of acquired IM resistance is a critical issue. We explored the mechanisms which influence IM efficacy and resistance in CML. Higher protective autophagy was identified in IM-resistant K562 (K562R) cells. Inhibition of autophagy by the inhibitors, chloroquine and 3-methyladenine, enhanced IM's efficacy in K562R cells. In addition, microRNA (miR)-199a/b-5p were downregulated in K562R cells compared to parent cells. Overexpression of miR-199a/b-5p reduced autophagy and induced cell apoptosis, resulting in enhanced IM's efficacy in K562R cells. Moreover, expression levels of the Wingless-type MMTV integration site family member 2 (WNT2), a positive regulator of autophagy, were significantly higher in K562R cells, and it was validated as a direct target gene of miR-199a/b-5p. Overexpressions of miR-199a/b-5p inhibited WNT2 downstream signaling. Furthermore, overexpression and knockdown of WNT2 influenced autophagy formation and CML drug sensitivity to IM. Overexpression of WNT2 could also reverse miR-199a/b-5p-enhanced IM efficacy in K562R cells. These results emphasized that miR-199a/b-5p inhibited autophagy via repressing WNT2 signaling and might provide novel therapeutic strategies for future IM-resistant CML therapy and drug development.

Identification of IGF-1-enhanced cytokine expressions targeted by miR-181d in glioblastomas via an integrative miRNA/mRNA regulatory network analysis.

The insulin-like growth factor (IGF)-1 signaling is relevant in regulating cell growth and cytokine secretions by glioblastomas. MicroRNAs determine the cell fate in glioblastomas. However, relationships between IGF-1 signaling and miRNAs in glioblastoma pathogenesis are still unclear. Our aim was to validate the IGF-1-mediated mRNA/miRNA regulatory network in glioblastomas. Using in silico analyses of mRNA array and RNA sequencing data from The Cancer Genome Atlas (TCGA), we identified 32 core enrichment genes that were highly associated with IGF-1-promoted cytokine-cytokine receptor interactions. To investigate the IGF-1-downregulated miRNA signature, microarray-based approaches with IGF-1-treated U87-MG cells and array data in TCGA were used. Four miRNAs, including microRNA (miR)-9-5p, miR-9-3p, miR-181d, and miR-130b, exhibited an inverse correlation with IGF-1 levels. The miR-181d, that targeted the most IGF-1-related cytokine genes, was significantly reduced in IGF-1-treated glioma cells. Statistical models incorporating both high-IGF-1 and low-miR-181d statuses better predicted poor patient survival, and can be used as an independent prognostic factor in glioblastomas. The C-C chemokine receptor type 1 (CCR1) and interleukin (IL)-1b demonstrated inverse correlations with miR-181d levels and associations with patient survival. miR-181d significantly attenuated IGF-1-upregulated CCR1 and IL-1b gene expressions. These findings demonstrate a distinct role for IGF-1 signaling in glioma progression via miR-181d/cytokine networks.

The microRNA-302b-inhibited insulin-like growth factor-binding protein 2 signaling pathway induces glioma cell apoptosis by targeting nuclear factor IA.

MicroRNAs are small noncoding RNAs that post-transcriptionally control the expression of genes involved in glioblastoma multiforme (GBM) development. Although miR-302b functions as a tumor suppressor, its role in GBM is still unclear. Therefore, this study comprehensively explored the roles of miR-302b-mediated gene networks in GBM cell death. We found that miR-302b levels were significantly higher in primary astrocytes than in GBM cell lines. miR-302b overexpression dose dependently reduced U87-MG cell viability and induced apoptosis through caspase-3 activation and poly(ADP ribose) polymerase degradation. A transcriptome microarray revealed 150 downregulated genes and 380 upregulated genes in miR-302b-overexpressing cells. Nuclear factor IA (NFIA), higher levels of which were significantly related to poor survival, was identified as a direct target gene of miR-302b and was involved in miR-302b-induced glioma cell death. Higher NFIA levels were observed in GBM cell lines and human tumor sections compared with astrocytes and non-tumor tissues, respectively. NFIA knockdown significantly enhanced apoptosis. We found high levels of insulin-like growth factor-binding protein 2 (IGFBP2), another miR-302b-downregulated gene, in patients with poor survival. We verified that NFIA binds to the IGFBP2 promoter and transcriptionally enhances IGFBP2 expression levels. We identified that NFIA-mediated IGFBP2 signaling pathways are involved in miR-302b-induced glioma cell death. The identification of a regulatory loop whereby miR-302b inhibits NFIA, leading to a decrease in expression of IGFBP-2, may provide novel directions for developing therapies to target glioblastoma tumorigenesis.

Correction: MicroRNA-1301-Mediated RanGAP1 Downregulation Induces BCR-ABL Nuclear Entrapment to Enhance Imatinib Efficacy in Chronic Myeloid Leukemia Cells.

[This corrects the article DOI: 10.1371/journal.pone.0156260.].

H2O2-Depleting and O2-Generating Selenium Nanoparticles for Fluorescence Imaging and Photodynamic Treatment of Proinflammatory-Activated Macrophages.

Macrophages have a pivotal role in chronic inflammatory diseases (CIDs), so imaging and controlling activated macrophage is critical for detecting and reducing chronic inflammation. In this study, photodynamic selenium nanoparticles (SeNPs) with photosensitive and macrophage-targeting bilayers were developed. The first layer of the photosensitive macromolecule was composed of a conjugate of a photosensitizer (rose bengal, RB) and a thiolated chitosan (chitosan-glutathione), resulting in a plasmonic coupling-induced red shift and broadening of RB absorption bands with increased absorption intensity. Electron paramagnetic resonance (EPR) and diphenylanthracene (DPA) quenching studies revealed that the SeNPs that were coated with the photosensitive layer were more effective than RB alone in producing singlet oxygen (1O2) under photoirradiation. The second layer of the activated macrophage-targetable macromolecule was synthesized by conjugation of hyaluronic acid with folic acid using an ethylenediamine linker. Proinflammatory-activated macrophages rapidly internalized the SeNPs that were covered with the targeting ligand, exhibiting a much stronger fluorescence signal of the SeNPs than did the nonactivated macrophages. Since proinflammatory-activated macrophage was known to generate a substantial amount of H2O2 while the inflamed site generally caused inflammation-associated tissue hypoxia, the SeNPs were further modified with O2 self-sufficient function for photodynamic therapy. Catalase was immobilized on the SeNPs by the formation of disulfide bonds. Intracellular reduction of disulfide bonds induced the subsequent release of catalase, which catalyzed the decomposition of H2O2. The H2O2-depleting and O2-generating photodynamic SeNPs efficiently killed activated macrophages and quenched the intracellular H2O2 and NO that are associated with inflammation. The SeNPs may have potential as a theranostic nanomaterial to image and control the activation of macrophages.

The CHAC1-inhibited Notch3 pathway is involved in temozolomide-induced glioma cytotoxicity.

Glioblastoma multiforme (GBM) is the high-grade primary glioma in adults. Temozolomide (TMZ), an alkylating agent of the imidazotetrazine series, is a first-line chemotherapeutic drug for clinical therapy. However, the expense of TMZ therapy and increasing drug resistance to TMZ decreases its therapeutic effects. Therefore, our aim was to investigate the detailed molecular mechanisms of TMZ-mediated cytotoxicity to enhance the efficacy of TMZ in clinical GBM therapy. First, TMZ-mediated gene expression profiles and networks in U87-MG cells were identified by transcriptome microarray and bioinformatic analyses. Cation transport regulator-like protein 1 (CHAC1) was the most highly TMZ-upregulated gene. Overexpression and knockdown of CHAC1 expression significantly influenced TMZ-mediated cell viability, apoptosis, caspase-3 activation, and poly(ADP ribose) polymerase (PARP) degradation. The c-Jun N-terminal kinase (JNK)1/c-JUN pathway was identified to participate in TMZ-upregulated CHAC1 expression via transcriptional control. Furthermore, CHAC1 levels were significantly decreased in GBM cell lines, TCGA array data, and tumor tissues. Overexpression of CHAC1 enhanced glioma apoptotic death via caspase-3/9 activation, PARP degradation, autophagy formation, reactive oxygen species generation, increased intracellular calcium, and loss of the mitochondria membrane potential. Finally, we also identified that TMZ significantly reduced Notch3 levels, which are upregulated in gliomas. TMZ also induced CHAC1 to bind to the Notch3 protein and inhibit Notch3 activation, resulting in attenuation of Notch3-mediated downstream signaling pathways. These results emphasize that CHAC1-inhibited Notch3 signaling can influence TMZ-mediated cytotoxicity. Our findings may provide novel therapeutic strategies for future glioblastoma therapy.