O-GlcNAcylation of RAB10 promotes hepatocellular carcinoma progression.

Ras-related protein Rab-10 (RAB10) is involved in tumorigenesis and progression of hepatocellular carcinoma (HCC). Here, we found RAB10, O-GlcNAc transferase (OGT), and O-GlcNAcylation were upregulated in HCC. In addition, RAB10 protein level was prominently positively correlated with the expression of OGT. O-GlcNAcylation modification of RAB10 was then investigated. Here we showed that RAB10 interacts directly with OGT in HCC cell lines, Meanwhile, O-GlcNAcylation enhanced RAB10 protein stability. Furthermore, knockdown of OGT suppressed aggressive behaviors of HCC in vitro and in vivo, while elevated RAB10 reversed these. Taken together, these results indicated that OGT mediated O-GlcNAcylation stabilized RAB10, thus accelerating HCC progression.

[Clinical features of children with febrile seizures caused by Omicron variant infection]. / Omicronå˜å¼‚æ ªæ„ŸæŸ“å¯¼è‡´å„¿ç«¥çƒ­æ€§æƒŠåŽ¥çš„ä¸´åºŠç‰¹å¾åˆ†æž.

OBJECTIVES: To study the clinical features of children with febrile seizures after Omicron variant infection. METHODS: A retrospective analysis was performed on the clinical data of children with febrile seizures after Omicron variant infection who were admitted to the Department of Neurology, Children's Hospital Affiliated to the Capital Institute of Pediatrics, from December 1 to 31, 2022 (during the epidemic of Omicron variant; Omicron group), and the children with febrile seizures (without Omicron variant infection) who were admitted from December 1 to 31, in 2021 were included as the non-Omicron group. Clinical features were compared between the two groups. RESULTS: There were 381 children in the Omicron group (250 boys and 131 girls), with a mean age of (3.2±2.4) years. There were 112 children in the non-Omicron group (72 boys and 40 girls), with a mean age of (3.5±1.8) years. The number of children in the Omicron group was 3.4 times that in the non-Omicron group. The proportion of children in two age groups, aged 1 to <2 years and 6-10.83 years, in the Omicron group was higher than that in the non-Omicron group, while the proportion of children in two age groups, aged 4 to <5 years and 5 to <6 years, was lower in the Omicron group than that in the non-Omicron group (P<0.05).The Omicron group had a significantly higher proportion of children with cluster seizures and status convulsion than the non-Omicron group (P<0.05). Among the children with recurrence of febrile seizures, the proportion of children aged 6-10.83 years in the Omicron group was higher than that in the non-Omicron group, while the proportion of children aged 3 years, 4 years, and 5 years in the Omicron group was lower than that in the non-Omicron group (P<0.05). CONCLUSIONS: Children with febrile seizures after Omicron variant infection tend to have a wider age range, with an increase in the proportion of children with cluster seizures and status convulsion during the course of fever.

Inhibition of NOS1 promotes the interferon response of melanoma cells.

BACKGROUND: NOS1 expression predicts poor prognosis in patients with melanoma. However, the molecular function of NOS1 in the type I IFN response and immune escape of melanoma is still unknown. METHODS: The CRISPR/Cas9 system was used to generate NOS1-knockout melanoma cells and the biological characteristics of NOS1-knockout cells were evaluated by MTT assay, clonogenic assay, EdU assay, and flow cytometric assay. The effect on tumor growth was tested in BALB/c-nu and C57BL/6 mouse models. The gene expression profiles were detected with Affymetrix microarray and RNA-seq and KEGG (Kyoto Encyclopedia of Genes and Genomes) and CLUE GO analysis was done. The clinical data and transcriptional profiles of melanoma patients from the public database TCGA (The Cancer Genome Atlas) and GEO (Gene Expression Omnibus, GSE32611) were analyzed by Qlucore Omics Explorer. RESULTS: NOS1 deletion suppressed the proliferation of melanoma A375 cells in culture, blocked cell cycling at the G0/G1 phase, and decreased the tumor growth in lung metastasis nodes in a B16 melanoma xenograft mouse model. Moreover, NOS1 knockout increased the infiltration of CD3+ immune cells in tumors. The transcriptomics analysis identified 2203 differential expression genes (DEGs) after NOS1 deletion. These DEGs indicated that NOS1 deletion downregulated mostly metabolic functions but upregulated immune response pathways. After inhibiting with NOS1 inhibitor N-PLA, melanoma cells significantly increased the response to IFN[Formula: see text] by upregulation expression of IFN[Formula: see text] simulation genes (ISGs), especially the components in innate immune signaling, JAK-STAT, and TOLL-LIKE pathway. Furthermore, these NOS1-regulating immune genes (NOS1-ISGs) worked as a signature to predict poor overall survival and lower response to chemotherapy in melanoma patients. CONCLUSION: These findings provided a transcriptional evidence of NOS1 promotion on tumor growth, which is correlated with metabolic regulation and immune escape in melanoma cells.

Should corticosteroids be administered for local infiltration analgesia in knee arthroplasty? A meta-analysis and systematic review.

WHAT IS KNOWN AND OBJECTIVE: The benefits of local infiltration analgesia (LIA) in knee arthroplasty (KA) have been well-documented. However, it is unknown whether adding a corticosteroid to the composition of the LIA is beneficial. This study aimed to investigate the efficacy and safety of administering periarticular steroids intraoperatively in patients who underwent KA through a systematic review and meta-analysis. METHODS: A systematic search was conducted to identify relevant randomized controlled trials in the PubMed, Embase, Web of Science and Cochrane databases up to January 19th, 2021 to perform a meta-analysis. Outcome variables included pain scores, total opioid consumption, knee range of motion (ROM) and postoperative complications. RESULTS: Corticosteroid injections did not reduce pain scores at 6, 12, 24 or 72 h postoperatively, although a minimal degree of transient pain relief was achieved at 48 h postoperatively compared with those in the placebo group, nor was there a significant difference in total opioid consumption. However, patients receiving corticosteroids did exhibit a transient ROM increase on postoperative days 1, 2 and 3. Since the minimal clinically important difference (MCID) for ROM is unclear, it is unknown if the improvement in ROM is clinically significant. WHAT IS NEW AND CONCLUSION: Our specific end-point analysis demonstrated that corticosteroid administration did not provide pain relief or reduce opioid consumption compared with placebo. However, corticosteroids might provide a statistically significant, though transient and minimal improvement in knee ROM after KA, although no firm conclusions about the benefits of administering corticosteroids in KA can be made based on the available evidence.

Microcystins-LR induced apoptosis via S-nitrosylation of GAPDH in colorectal cancer cells.

Microcystins-LR (MC-LR), a cyanobacterial toxins, initiate apoptosis in normal and tumor cells. Nitric oxide produced by iNOS is necessary for MC-LR-induced apoptosis. However, the underlying mechanism of NO mediated MC-LR cytotoxicity remains unclear. Here, we performed in vitro experiments on MC-LR cytotoxicity associated with NO induced S-nitrosyation of GAPDH in human colon cancer cells SW480. MTT assay indicated that MC-LR decreased the cellular viability by high concentration (>1 µM). Flow cytometer assay revealed that apoptosis was core mode for MC-LR cytotoxicity. Griess assay showed that MC-LR exposure increased the release of NO through the function of NOS1 and NOS2 in SW480 cells. In turn, NO stress induced the S-nitrosylated modification of GAPDH leading to its nuclear translocation following Siah1 binding. CHIP assay showed that the nuclear GADPH increased P53 transcript of a panner of apoptosis related genes. Moreover, apoptosis induced by MC-LR could be reduced by GAPDH or si-Siah1 or NOSs inhibitor, L-NAME. Thus, our study verified a molecular mechanism of NO/GAPDH/Siah1 cascade in MC-LR mediated apoptosis in colorectal cancer cells, providing a further understanding the in vitro molecular mechanism of MC-LR colorectal toxicity.

Mitochondrial NOS1 suppresses apoptosis in colon cancer cells through increasing SIRT3 activity.

Previous studies have suggested that nitric oxide (NO) which is synthetized by nitric oxide synthase (NOS) is closely related to the carcinogenesis and progression of colon cancer. However, the precise physiopathological role of NO on colon cancer remains unclear, and a lot of related studies focused on NOS2 and NOS3, but little on NOS1. Here, stable overexpression NOS1 of colon cancer cells were constructed to investigate whether NOS1 plays a special role in colon cancer. We observed that NOS1 protein was presented in mitochondria. Both the basal and cisplatin-induced mitochondrial superoxide were inhibited by NOS1, and the cisplatin-induced apoptosis was also inhibited by NOS1. Geldanamycin, a Hsp90 N-terminal inhibitor, was able to impede NOS1 translocation into mitochondria and reverse NOS1-induced apoptosis resistance. Importantly, SIRT3 activity was enhanced by NOS1, which contributes to the low level of mitochondrial superoxide and apoptosis resistance. Our data suggest a link between NOS1 and apoptosis resistance in colon cancer cells through mtNOS1-SIRT3-SOD2 axis. Furthermore, NOS1-induced apoptosis resistance could be reversed by inhibiting mitochondrial translocation of NOS1.

Associations between maternal BMI as well as glucose tolerance and adverse pregnancy outcomes in women with polycystic ovary syndrome.

This retrospective, cohort study examined the association between maternal pre-pregnancy body mass index (BMI), independent of glucose tolerance and adverse pregnancy outcomes in women with polycystic ovary syndrome (PCOS), for which there are few previous studies. Medical records from 2012 to 2015 at Guangzhou Women and Children's Medical Center, China were reviewed for women previously diagnosed with PCOS with normal 2-h 75-g oral glucose tolerance test (OGTT) results (n = 1249). The separate and joint effects of maternal BMI and glucose levels on pregnancy outcomes were assessed. Maternal pre-pregnancy BMI was associated with hypertensive disorders of pregnancy (HDP) (OR: 1.22, 95% CI: 1.02-1.45), preterm birth (OR: 1.49, 95% CI: 1.08-2.17), and large for gestational age (LGA) (OR: 1.69, 95% CI: 1.16-2.20). Elevated fasting glucose and maternal pre-pregnancy BMI were jointly associated with increased risks of HDP, preterm birth, and LGA. Therefore, among women with PCOS and normal glucose tolerance, maternal pre-pregnancy BMI is an independent risk factor of adverse pregnancy outcomes.

AKT-mediated regulation of chromatin ubiquitylation and tumorigenesis through Mel18 phosphorylation.

Polycomb repressor complex 1 (PRC1) is linked to the regulation of gene expression and histone ubiquitylation conformation, which contributes to carcinogenesis. However, the upstream regulators of PRC1 biogenesis machinery remain obscure. Here, we report that the polycomb group-related mammalian gene Mel18 is a target of the protein kinase AKT. AKT phosphorylates Mel18 at T334 to disrupt the interaction between Mel18 and other PRC1 members, leading to attenuated PRC1-dependent ubiquitylation of histone H2A at Lys119. As such, PRC1 target genes, many of which are known oncogenes, are derepressed upon T334-Mel18 phosphorylation, which promotes malignant behaviours, including cell proliferation, tumour formation, migration and invasion, bone and brain metastatic lesion formation. Notably, a positive correlation between AKT activity and pT334-Mel18 is observed, and prognostic models based on p-AKT and pT334-Mel18 that predicted overall survival and distant metastasis-free survival in breast cancer patients are established. These findings have implications for understanding the role of AKT and its associated proteins in chromatin ubiquitylation, and also indicate the AKT-Mel18-H2AK119ub axis as a novel prognostic biomarker and therapeutic target for cancer patients.

Fis1 phosphorylation by Met promotes mitochondrial fission and hepatocellular carcinoma metastasis.

Met tyrosine kinase, a receptor for a hepatocyte growth factor (HGF), plays a critical role in tumor growth, metastasis, and drug resistance. Mitochondria are highly dynamic and undergo fission and fusion to maintain a functional mitochondrial network. Dysregulated mitochondrial dynamics are responsible for the progression and metastasis of many cancers. Here, using structured illumination microscopy (SIM) and high spatial and temporal resolution live cell imaging, we identified mitochondrial trafficking of receptor tyrosine kinase Met. The contacts between activated Met kinase and mitochondria formed dramatically, and an intact HGF/Met axis was necessary for dysregulated mitochondrial fission and cancer cell movements. Mechanically, we found that Met directly phosphorylated outer mitochondrial membrane protein Fis1 at Tyr38 (Fis1 pY38). Fis1 pY38 promoted mitochondrial fission by recruiting the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) to mitochondria. Fragmented mitochondria fueled actin filament remodeling and lamellipodia or invadopodia formation to facilitate cell metastasis in hepatocellular carcinoma (HCC) cells both in vitro and in vivo. These findings reveal a novel and noncanonical pathway of Met receptor tyrosine kinase in the regulation of mitochondrial activities, which may provide a therapeutic target for metastatic HCC.

NOS1 expression promotes proliferation and invasion and enhances chemoresistance in ovarian cancer.

Nitric oxide (NO), an important chemical messenger, serves a dual role in tumor progression. Nitric oxide synthase isoform 1 (NOS1) was observed to be increasingly expressed in various types of cancer, and its expression has been associated with tumor progression. However, the level of NOS1 expression and the associated functions of NOS1 in human ovarian cancer remain undefined. Using gene expression profiles of ovarian cancer from the Gene Expression Omnibus (GEO) database, the present study revealed that NOS1 was increasingly expressed in ovarian cancer tissues. The present study investigated the level of NOS1 expression and its effects on in vitro cell function, including proliferation, migration and invasion as well as chemoresistance to cispatin (DDP) treatment in OVCAR3 cells. Reverse transcription-quantitative polymerase chain reaction demonstrated that the level of NOS1 mRNA expression varied in different ovarian cancer lines. However, immunoblotting indicated that the level of NOS1 protein expression was constitutively high in ovarian cancer cell lines. Treatment with NOS inhibitor NG-nitro-L-arginine methyl ester or transfection with NOS1 short hairpin RNA significantly inhibited cell proliferation, migration and invasion compared with the control, whereas the sensitivity of OVCAR3 cells to DDP treatment was increased. The results of the present study indicated that NOS1 promoted the function of ovarian cancer cells, including proliferation, invasion and chemoresistance, providing a potential target for ovarian cancer therapeutic.

The heterocyclic compound Tempol inhibits the growth of cancer cells by interfering with glutamine metabolism.

Tempol (4-hydroxy-2,2,6,6-Tetramethylpiperidine-1-oxyl, TPL), a nitroxide compound, inhibits proliferation and increases the vulnerability of cancer cells to apoptosis induced by cytotoxic agents. However, the molecular mechanism of TPL inhibiting cancer cell proliferation has not been fully understood. In this study, we evaluated the metabolic effect of TPL on cancer cells and explored its cancer therapeutic potential. Extracellular flow assays showed that TPL inhibited cellular basal and maximal oxygen consumption rates of mitochondrial. 13C metabolic flux analysis showed that TPL treatment had minimal effect on glycolysis. However, we found that TPL inhibits glutamine metabolism by interfering with the oxidative tricarboxylic acid cycle (TCA) process and reductive glutamine process. We found that the inhibitory effect of TPL on metabolism occurs mainly on the step from citrate to α-ketoglutarate or vice versa. We also found that activity of isocitrate dehydrogenase IDH1 and IDH2, the key enzymes in TCA, were inhibited by TPL treatment. In xenograft mouse model, TPL treatment reduced tumor growth by inhibiting cellular proliferation of xenograft tumors. Thus, we provided a mechanism of TPL inhibiting cancer cell proliferation by interfering with glutamine utilization that is important for survival and proliferation of cancer cells. The study may help the development of a therapeutic strategy of TPL combined with other anticancer medicines.

Publisher Correction: NOS1 S-nitrosylates PTEN and inhibits autophagy in nasopharyngeal carcinoma cells.

It was brought to the attention of the Editors that there had been an accidental duplication of the western blot images during the preparation of Figs. 1b and c. The authors were notified about the error and have supplied the correct image for Fig. 1c (below). We apologize for any inconvenience this may have caused readers.

NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2.

BACKGROUND: The dysfunction of type I interferon (IFN) signaling is an important mechanism of immune escape and metastasis in tumors. Increased NOS1 expression has been detected in melanoma, which correlated with dysfunctional IFN signaling and poor response to immunotherapy, but the specific mechanism has not been determined. In this study, we investigated the regulation of NOS1 on the interferon response and clarified the relevant molecular mechanisms. METHODS: After stable transfection of A375 cells with NOS1 expression plasmids, the transcription and expression of IFNα-stimulated genes (ISGs) were assessed using pISRE luciferase reporter gene analysis, RT-PCR, and western blotting, respectively. The effect of NOS1 on lung metastasis was assessed in melanoma mouse models. A biotin-switch assay was performed to detect the S-nitrosylation of HDAC2 by NOS1. ChIP-qPCR was conducted to measure the binding of HDAC2, H4K16ac, H4K5ac, H3ac, and RNA polymerase II in the promoters of ISGs after IFNα stimulation. This effect was further evaluated by altering the expression level of HDAC2 or by transfecting the HDAC2-C262A/C274A site mutant plasmids into cells. The coimmunoprecipitation assay was performed to detect the interaction of HDAC2 with STAT1 and STAT2. Loss-of-function and gain-of-function approaches were used to examine the effect of HDAC2-C262A/C274A on lung metastasis. Tumor infiltrating lymphocytes were analyzed by flow cytometry. RESULTS: HDAC2 is recruited to the promoter of ISGs and deacetylates H4K16 for the optimal expression of ISGs in response to IFNα treatment. Overexpression of NOS1 in melanoma cells decreases IFNα-responsiveness and induces the S-nitrosylation of HDAC2-C262/C274. This modification decreases the binding of HDAC2 with STAT1, thereby reducing the recruitment of HDAC2 to the ISG promoter and the deacetylation of H4K16. Moreover, expression of a mutant form of HDAC2, which cannot be nitrosylated, reverses the inhibition of ISG expression by NOS1 in vitro and decreases NOS1-induced lung metastasis and inhibition of tumor infiltrating lymphocytes in a melanoma mouse model. CONCLUSIONS: This study provides evidence that NOS1 induces dysfunctional IFN signaling to promote lung metastasis in melanoma, highlighting NOS1-induced S-nitrosylation of HDAC2 in the regulation of IFN signaling via histone modification.

Polo-Like Kinase 1 phosphorylates and stabilizes KLF4 to promote tumorigenesis in nasopharyngeal carcinoma.

Rationale: Advanced nasopharyngeal carcinoma (NPC) is an aggressive disease with no targeted therapies and poor outcomes. New innovative targets are urgently needed. KLF4 has been extensively studied in the context of tumors, and current data suggest that it can act as either a tissue-specific tumor-inhibiting or a tumor-promoting gene. Here, we found that KLF4 played as a tumor-promoting gene in NPC, and could be mediated by PLK1. Methods: Tissue immunohistochemistry (IHC) assay was performed to identify the role of KLF4 in NPC. Global gene expression experiments were performed to explore the molecular mechanisms underlying KLF4-dependent tumorigenesis. Small-molecule kinase inhibitor screening was performed to identify potential upstream kinases of KLF4. The pharmacologic activity of polo-like kinase inhibitor volasertib (BI6727) in vitro and in vivo was determined. Result: Our investigation showed that high expression of KLF4 was correlated with poor prognosis in NPC. Moreover, genome-wide profiling revealed that KLF4 directly activated oncogenic programmes, including gene sets associated with KRAS, VEGF, and MYC signalling. We further found that inhibition of polo-like kinase 1 could downregulate the expression of KLF4 and that PLK1 directly phosphorylated KLF4 at Ser234. Notably, phosphorylation of KLF4 by PLK1 caused the recruitment and binding of the E3 ligase TRAF6, which resulted in KLF4 K32 K63-linked ubiquitination and stabilization. Moreover, KLF4 could enhance TRAF6 expression at the transcriptional level, thus initiating a KLF4-TRAF6 feed-forward loop. Treatment with the PLK1 inhibitor volasertib (BI6727) significantly inhibited tumor growth in nude mice. Conclusion: Our study unveiled a new PLK1-TRAF6-KLF4 feed-forward loop. The resulting increase in KLF4 ubiquitination leads to stabilization and upregulation of KLF4, which leads to tumorigenesis in NPC. These results expand our understanding of the role of KLF4 in NPC and validate PLK1 inhibitors as potential therapeutic agents for NPC, especially cancer patients with KLF4 overexpression.

iNOS-derived nitric oxide promotes glycolysis by inducing pyruvate kinase M2 nuclear translocation in ovarian cancer.

Aerobic glycolysis is essential for tumor growth and survival. Activation of multiple carcinogenic signals contributes to metabolism reprogramming during malignant transformation of cancer. Recently nitric oxide has been noted to promote glycolysis but the mechanism remains elusive. We report here the dual role of nitric oxide in glycolysis: low/physiological nitric oxide (≤ 100 nM) promotes glycolysis for ATP production, oxidative defense and cell proliferation of ovary cancer cells, whereas excess nitric oxide (≥ 500 nM) inhibits it. Nitric oxide has a positive effect on glycolysis by inducing PKM2 nuclear translocation in an EGFR/ERK2 signaling-dependent manner. Moreover, iNOS induced by mild inflammatory stimulation increased glycolysis and cell proliferation by producing low doses of nitric oxide, while hyper inflammation induced iNOS inhibited it by producing excess nitric oxide. Finally, iNOS expression is abnormally increased in ovarian cancer tissues and is correlated with PKM2 expression. Overexpression of iNOS is associated with aggressive phenotype and poor survival outcome in ovarian cancer patients. Our study indicated that iNOS/NO play a dual role of in tumor glycolysis and progression, and established a bridge between iNOS/NO signaling pathway and EGFR/ERK2/PKM2 signaling pathway, suggesting that interfering glycolysis by targeting the iNOS/NO/PKM2 axis may be a valuable new therapeutic approach of treating ovarian cancer.

NOS1 S-nitrosylates PTEN and inhibits autophagy in nasopharyngeal carcinoma cells.

Autophagy is a cellular survival mechanism that involves the catabolic degradation of damaged proteins and organelles during periods of metabolic stress, and when overly stimulated, commonly contributes to cell death. Nitric oxide (NO), a potent cellular messenger, participates in a complex mechanism which assists in controlling autophagy. However, the mechanism by which endogenous NO formed by distinct isoforms of nitric oxide synthase (NOS) helps to regulate autophagy in cancer cells remains unclear. Here we report that NOS1 reduces excessive levels of autophagy and promotes the survival of nasopharyngeal carcinoma cells. We found that inhibition of NOS1 increased cell death resulting from siRNA or the use of pharmacologic agents; and this effect was reversed by the autophagy inhibitor, chloroquine. The role of NOS1 in the autophagy process depended on the activation of AKT/mTOR signaling by S-nitrosylation of phosphatase and tensin homolog (PTEN) proteins. The mechanism by which NOS1 modifies PTEN protein might involve a direct interaction between these two molecules. Moreover, in an in vivo study, the NOS1 inhibitor N(G)-nitro-L-arginine methyl ester activated AKT/mTOR signaling and promoted autophagy in xenograph tumors. Our studies demonstrated that NOS1 prevents excessive autophagy via S-nitrosylation of PTEN, and activation of the AKT/mTOR signaling pathway. PTEN and the AKT/mTOR signaling pathway are promising targets for improving the chemotherapeutic treatment of cancer.