Reprogramming of arachidonate metabolism confers temozolomide resistance to glioblastoma through enhancing mitochondrial activity in fatty acid oxidation.

BACKGROUND: Sp1 is involved in the recurrence of glioblastoma (GBM) due to the acquirement of resistance to temozolomide (TMZ). Particularly, the role of Sp1 in metabolic reprogramming for drug resistance remains unknown. METHODS: RNA-Seq and mass spectrometry were used to analyze gene expression and metabolites amounts in paired GBM specimens (primary vs. recurrent) and in paired GBM cells (sensitive vs. resistant). ω-3/6 fatty acid and arachidonic acid (AA) metabolism in GBM patients were analyzed by targeted metabolome. Mitochondrial functions were determined by Seahorse XF Mito Stress Test, RNA-Seq, metabolome and substrate utilization for producing ATP. Therapeutic options targeting prostaglandin (PG) E2 in TMZ-resistant GBM were validated in vitro and in vivo. RESULTS: Among the metabolic pathways, Sp1 increased the prostaglandin-endoperoxide synthase 2 expression and PGE2 production in TMZ-resistant GBM. Mitochondrial genes and metabolites were obviously increased by PGE2, and these characteristics were required for developing resistance in GBM cells. For inducing TMZ resistance, PGE2 activated mitochondrial functions, including fatty acid ß-oxidation (FAO) and tricarboxylic acid (TCA) cycle progression, through PGE2 receptors, E-type prostanoid (EP)1 and EP3. Additionally, EP1 antagonist ONO-8713 inhibited the survival of TMZ-resistant GBM synergistically with TMZ. CONCLUSION: Sp1-regulated PGE2 production activates FAO and TCA cycle in mitochondria, through EP1 and EP3 receptors, resulting in TMZ resistance in GBM. These results will provide us a new strategy to attenuate drug resistance or to re-sensitize recurred GBM.

Glioma-targeted delivery of exosome-encapsulated antisense oligonucleotides using neural stem cells.

Tropism of neural stem cells (NSCs) to hypoxic tumor areas provides an opportunity for the drug delivery. Here, we demonstrate that NSCs effectively transport antisense oligonucleotides (ASOs) targeting oncogenic and tolerogenic signal transducer and activator of transcription 3 (STAT3) protein into glioma microenvironment. To enable spontaneous, scavenger receptor-mediated endocytosis by NSCs, we used previously described CpG-STAT3ASO conjugates. Following uptake and endosomal escape, CpG-STAT3ASO colocalized with CD63+ vesicles and later with CD63+CD81+ exosomes. Over 3 days, NSCs secreted exosomes loaded up to 80% with CpG-STAT3ASO. Compared to native NSC exosomes, the CpG-STAT3ASO-loaded exosomes potently stimulated immune activity of human dendritic cells or mouse macrophages, inducing nuclear factor κB (NF-κB) signaling and interleukin-12 (IL-12) production. Using orthotopic GL261 tumors, we confirmed that NSC-mediated delivery improved oligonucleotide transfer from a distant injection site into the glioma microenvironment versus naked oligonucleotides. Correspondingly, the NSC-delivered CpG-STAT3ASO enhanced activation of glioma-associated microglia. Finally, we demonstrated that NSC-mediated CpG-STAT3ASO delivery resulted in enhanced antitumor effects against GL261 glioma in mice. Peritumoral injections of 5 × 105 NSCs loaded ex vivo with CpG-STAT3ASO inhibited subcutaneous tumor growth more effectively than the equivalent amount of oligonucleotide alone. Based on these results, we anticipate that NSCs and NSC-derived exosomes will provide a clinically relevant strategy to improve delivery and safety of oligonucleotide therapeutics for glioma treatment.

17ß-estradiol induces temozolomide resistance through NRF2-mediated redox homeostasis in glioblastoma.

Glioblastoma multiforme (GBM) is the most fatal cancer among brain tumors, and the standard treatment of GBM patients is surgical tumor resection followed by radiotherapy and temozolomide (TMZ) chemotherapy. However, tumors always recur due to the developing drug resistance. It has been shown that neurosteroids, including dehydroepiandrosterone and 17ß-estradiol, are synthesized in TMZ-resistant GBM tumors. Therefore, we sought to explore the possible role of 17ß-estradiol in the development of drug resistance in GBM. Bioinformatics analysis revealed that aromatase/cytochrome P450 19A1 expression was gradually increased in the development from normal, astrocytoma to GBM. The level of 17ß-estradiol was significantly increased in TMZ-resistant cells characterized by ultra performance liquid chromatography-tandem mass spectrometry. Furthermore, 17ß-estradiol attenuated TMZ-induced cell death and reduced reactive oxygen species production by mitochondria. In addition, 17ß-estradiol attenuated oxidative stress by increasing the expression of superoxide dismutase 1/2, catalase, and nuclear factor erythroid 2-related factor (NRF) 2. We found that NRF2 expression was essential for the induction of drug resistance by 17ß-estradiol through the reduction of oxidative stress in GBM.

Sp1 in Astrocyte Is Important for Neurite Outgrowth and Synaptogenesis.

In this study, we found that Sp1 was highly expressed in astrocytes, implying that Sp1 might be important for the function of astrocytes. Sp1/GFAP-Cre-ERT2 conditional knockout mice were constructed to study the role of Sp1 in astrocytes. Knockout of Sp1 in astrocytes altered astrocytic morphology and decreased GFAP expression in the cortex and hippocampus but did not affect cell viability. Loss of Sp1 in astrocytes decreased the number of neurons in the cortex and hippocampus. Conditioned medium from primary astrocytes with Sp1 knockout disrupted neuronal dendritic outgrowth and synapse formation, resulting in abnormal learning, memory, and motor behavior. Sp1 knockout in astrocytes altered gene expression, including decreasing the expression of Toll-like receptor 2 and Cfb and increasing the expression of C1q and C4Bp, thereby affecting neurite outgrowth and synapse formation, resulting in disordered neuron function. Studying these gene regulations might be beneficial to understanding neuronal development and brain injury prevention.

Correction: A novel derivative of betulinic acid, SYK023, suppresses lung cancer growth and malignancy.

[This corrects the article DOI: 10.18632/oncotarget.3701.].

USP24 induces IL-6 in tumor-associated microenvironment by stabilizing p300 and ß-TrCP and promotes cancer malignancy.

We have previously demonstrated that USP24 is involved in cancer progression. Here, we found that USP24 expression is upregulated in M2 macrophages and lung cancer cells. Conditioned medium from USP24-knockdown M2 macrophages decreases the migratory and chemotactic activity of lung cancer cells and the angiogenic properties of human microvascular endothelial cell 1 (HMEC-1). IL-6 expression is significantly decreased in USP24-knockdown M2 macrophages and lung cancer cells, and IL-6-replenished conditioned medium restores the migratory, chemotactic and angiogenetic properties of the cells. USP24 stabilizes p300 and ß-TrCP to increase the levels of histone-3 acetylation and NF-κB, and decreases the levels of DNMT1 and IκB, thereby increasing IL-6 transcription in M2 macrophages and lung cancer cells, results in cancer malignancy finally. IL-6 has previously been a target for cancer drug development. Here, we provide direct evidence to support that USP24 promotes IL-6 expression, which might be beneficial for cancer therapy.

Melatonin protects brain against ischemia/reperfusion injury by attenuating endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress plays a vital role in mediating ischemic reperfusion damage in brain. In this study, we evaluated whether melatonin inhibits ER stress in cultured neurons exposed to oxygen and glucose deprivation (OGD) and in rats subjected to transient focal cerebral ischemia. Sprague-Dawley rats were treated with melatonin (5 mg/kg) or control at reperfusion onset after transient occlusion of the right middle cerebral artery (MCA) for 90 min. Brain infarction and hemorrhage within infarcts were measured. The expression of ER stress proteins of phosphorylation of PRKR­like endoplasmic reticulum kinase (p-PERK), phosphorylation of eukaryotic translation initiation factor 2α (p-eIF2α), activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) were detected by western blotting and immunohistochemistry analysis. The terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method, cleaved caspase-3 and cytochrome c were used to investigate cell apoptosis in OGD-induced cultured neurons. Our results demonstrated that animals treated with melatonin had significantly reduced infarction volumes and individual cortical lesion sizes as well as increased numbers of surviving neurons. Melatonin can significantly modulate protein levels by decreasing both p-PERK and p-eIF2α in the ischemic core and penumbra. Moreover, the expressions of ATF4 and CHOP were restrained in the ischemic core and penumbra, respectively. Furthermore, pretreatment with melatonin at 10-100 µM effectively reduced the levels of p-PERK and p-eIF2α in cultured neurons after OGD injury. Melatonin treatment also effectively decreased neuron apoptosis resulting from OGD-induced neuron injury. These results indicate that melatonin effectively attenuated post-ischemic ER stress after ischemic stroke.

Nm23-H1-stabilized hnRNPA2/B1 promotes internal ribosomal entry site (IRES)-mediated translation of Sp1 in the lung cancer progression.

Our recent studies have indicated that specificity protein-1 (Sp1) accumulates substantially in the early stage of lung cancer but is partially decreased in the late stages, which is an important factor in the progression of the cancer. In this study, we found that Nm23-H1 and hnRNPA2/B1 could be recruited to the 5'UTR of Sp1 mRNA. In investigating the clinical relevance of Nm23-H1/Sp1 levels, we found a positive correlation between lung cancer patients with poor prognosis and low levels of Sp1 and Nm23-H1, suggesting an association between Nm23-H1/Sp1 levels and survival rate. Knockdown of Nm23-H1 inhibits lung cancer growth but increases lung cancer cell malignancy, which could be rescued by overexpression of Sp1, indicating that Nm23-H1-induced Sp1 expression is critical for lung cancer progression. We also found that Nm23-H1 increases the protein stability of hnRNPA2/B1and is thereby co-recruited to the 5'UTR of Sp1 mRNA to regulate cap-independent translational activity. Since the Sp1 level is tightly regulated during lung cancer progression, understanding the molecular mechanisms underlying the regulation by Nm23-H1/hnRNPA2B1 of Sp1 expression in the various stages of lung cancer will be beneficial for lung cancer therapy in the future.

Positive feedback regulation between IL10 and EGFR promotes lung cancer formation.

The role of IL10 in the tumorigenesis of various cancer types is still controversial. Here, we found that increased IL10 levels are correlated with a poor prognosis in lung cancer patients. Moreover, IL10 levels were significantly increased in the lungs and serum of EGFRL858R- and Kras4bG12D-induced lung cancer mice, indicating that IL10 might facilitate lung cancer tumorigenesis. IL10 knockout in EGFRL858R and Kras4bG12D mice inhibited the development of lung tumors and decreased the levels of infiltrating M2 macrophages and tumor-promoting Treg lymphocytes. We also showed that EGF increases IL10 expression by enhancing IL10 mRNA stability, and IL10 subsequently activates JAK1/STAT3, Src, PI3K/Akt, and Erk signaling pathways. Interestingly, the IL10-induced recruitment of phosphorylated Src was critical for inducing EGFR through the activation of the JAK1/STAT3 pathway, suggesting that Src and JAK1 positively regulate each other to enhance STAT3 activity. Doxycycline-induced EGFRL858R mice treated with gefitinib and anti-IL10 antibodies exhibited poor tumor formation. In conclusion, IL10 and EGFR regulate each other through positive feedback, which leads to lung cancer formation.

A novel derivative of betulinic acid, SYK023, suppresses lung cancer growth and malignancy.

Herein, we evaluated the anti-cancer effect and molecular mechanisms of a novel betulinic acid (BA) derivative, SYK023, by using two mouse models of lung cancer driven by KrasG12D or EGFRL858R. We found that SYK023 inhibits lung tumor proliferation, without side effects in vivo or cytotoxicity in primary lung cells in vitro. SYK023 triggered endoplasmic reticulum (ER) stress. Blockage of ER stress in SYK023-treated cells inhibited SYK023-induced apoptosis. In addition, we found that the expression of cell cycle-related genes, including cyclin A2, B1, D3, CDC25a, and CDC25b decreased but, while those of p15INK4b, p16INK4a, and p21CIP1 increased following SYK023 treatment. Finally, low doses of SYK023 significantly decreased lung cancer metastasis in vitro and in vivo. Expression of several genes related to cell migration, including synaptopodin, were downregulated by SYK023, thereby impairing F-actin polymerization and metastasis. Therefore, SYK023 may be a potentially therapeutic treatment for metastatic lung cancer.

Nucleolin enhances internal ribosomal entry site (IRES)-mediated translation of Sp1 in tumorigenesis.

Our previous study indicated that specificity protein-1 (Sp1) is accumulated during hypoxia in an internal ribosomal entry site (IRES)-dependent manner. Herein, we found that the Sp1 was induced strongly at the protein level, but not in the mRNA level, in lung tumor tissue, indicating that translational regulation might contribute to the Sp1 accumulation during tumorigenesis. A further study showed that the translation of Sp1 was dramatically induced through an IRES-dependent pathway. RNA immunoprecipitation analysis of proteins bound to the 5'-untranslated region (5'-UTR) of Sp1 identified interacting protein - nucleolin. Knockdown of nucleolin significantly inhibited IRES-mediated translation of Sp1, suggesting that nucleolin positively facilitates Sp1 IRES activation. Further analysis of the interaction between nucleolin and the 5'-UTR of Sp1 mRNA revealed that the GAR domain was important for IRES-mediated translation of Sp1. Moreover, gefitinib, and LY294002 and MK2206 compounds inhibited IRES-mediated Sp1 translation, implying that activation of the epithelial growth factor receptor (EGFR) pathway via Akt activation triggers the IRES pathway. In conclusion, EGFR activation-mediated nucleolin phosphorylated at Thr641 and Thr707 was recruited to the 5'-UTR of Sp1 as an IRES trans-acting factor to modulate Sp1 translation during lung cancer formation.

Phosphorylation of p300 increases its protein degradation to enhance the lung cancer progression.

p300 is a transcription cofactor for a number of nuclear proteins. Most studies of p300 have focused on the regulation of its function, which primarily includes its role as a transcription co-factor for a number of nuclear proteins. In this study, we found that p300 was highly phosphorylated and its level was decreased during mitosis and tumorigenesis. In vitro and in vivo experiments aimed showed that cyclin-dependent kinase 1 (CDK1) and ERK1/2 phosphorylated p300 on Ser1038 and Ser2039. Mutations of Ser1038 and Ser2039 increased p300 protein stability and levels. Inhibition of p300 degradation by blocking its phosphorylation decreased the proliferation and metastasis activity of lung cancer cells, indicating that p300 acts as a tumor suppressor in lung cancer tumorigenesis. Investigation of the molecular mechanism showed that blocking p300 phosphorylation disrupted chromatin condensation and the increased the acetylation of histone H3. Analysis of cell cycle progression in HA-p300-S2A-expressing cells by flow cytometry showed that the p300 mutants arrested the cells at S-phase or delayed the mitotic entry and exit. The expression of several important oncogenes, MMP-9, vimentin, ß-catenin, N-cadherin and c-myc, was negatively regulated by p300. In conclusion, during lung tumorigenesis, a phosphorylation-mediated decrease in p300 level enhanced oncogene expression during interphase and decreased histone H3 acetylation during mitosis, which promoted lung cancer progression.

Dry deposition of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in ambient air.

The PCDD/Fs in the ambient air associated with concentration and dry deposition flux of four seasons were characterized in rural area. The mean PCDD/F concentrations were 0.342, 0.221, 0.675, 0.741 pg m(-3) and the mean I-TEQ values were 0.027, 0.016, 0.024, 0.063 pg m(-3) in spring, summer, fall and winter, respectively. Ambient air in winter was higher by a factor of 3.4 and 3.9 for PCDD/F concentration and I-TEQ, respectively, than in summer. The study area is located in a Tropical region. Hence, domestic heating is not found in this area and is not responsible for the elevated winter concentration in comparison to other studies. A smooth plate with a sharp leading edge that is pointed into the wind by a wind vane was used for measuring dry deposition flux of PCDD/Fs. Atmospheric dry deposition fluxes of total PCDD/Fs were 140, 116, 137, and 207 pg m(-2)day(-1) in spring, summer, fall, and winter, respectively, and averaged approximately 150 pg m(-2)day(-1). The total dry deposition flux was found to decrease as the temperature increased. Calculated dry deposition velocities of total PCDD/Fs were 0.45, 0.52, 0.32 and 0.39 cm s(-1) in spring, summer, fall, and winter, respectively, and averaged 0.42 cm s(-1).