Chondroitin sulfate modification of CSPG4 regulates the maintenance and differentiation of glioma-initiating cells via integrin-associated signaling.

Glioma stem cell/glioma-initiating cell (GIC) and their niches are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanisms of GIC maintenance/differentiation, we performed a unique integrated proteogenomics utilizing GIC clones established from patient tumors having the potential to develop glioblastoma. After the integration and extraction of the transcriptomics/proteomics data, we found that chondroitin sulfate proteoglycan 4 (CSPG4) and its glycobiosynthetic enzymes were significantly upregulated in GICs. Glyco-quantitative PCR array revealed that chondroitin sulfate (CS) biosynthetic enzymes, such as xylosyltransferase 1 (XYLT1) and carbohydrate sulfotransferase 11, were significantly downregulated during serum-induced GIC differentiation. Simultaneously, the CS modification on CSPG4 was characteristically decreased during the differentiation and also downregulated by XYLT1 knockdown. Notably, the CS degradation on CSPG4 by ChondroitinaseABC treatment dramatically induced GIC differentiation, which was significantly inhibited by the addition of CS. GIC growth and differentiation ability were significantly suppressed by CSPG4 knockdown, suggesting that CS-CSPG4 is an important factor in GIC maintenance/differentiation. To understand the molecular function of CS-CSPG4, we analyzed its associating proteins in GICs and found that CSPG4, but not CS-CSPG4, interacts with integrin αV during GIC differentiation. This event sequentially upregulates integrin-extracellular signal-regulated kinase signaling, which can be inhibited by cyclic-RGD (Arg-Gly-Asp) integrin αV inhibitor. These results indicate that CS-CSPG4 regulates the GIC microenvironment for GIC maintenance/differentiation via the CS moiety, which controls integrin signaling. This study demonstrates a novel function of CS on CSPG4 as a niche factor, so-called "glyco-niche" for GICs, and suggests that CS-CSPG4 could be a potential target for malignant glioma.

Altered O-linked glycosylation in benign and malignant meningiomas.

Background: Changes in protein glycosylation have been reported in various diseases, including cancer; however, the consequences of altered glycosylation in meningiomas remains undefined. We established two benign meningioma cell lines-SUT-MG12 and SUT-MG14, WHO grade I-and demonstrated the glycan and glycosyltransferase profiles of the mucin-type O-linked glycosylation in the primary benign meningioma cells compared with two malignant meningioma cell lines-HKBMM and IOMM-Lee, WHO grade III. Changes in O-linked glycosylation profiles in malignant meningiomas were proposed. Methods: Primary culture technique, morphological analysis, and immunocytochemistry were used to establish and characterize two benign meningioma cell lines. The glycan profiles of the primary benign and malignant meningiomas cell lines were then analyzed using lectin cytochemistry. The gene expression of O-linked glycosyltransferases, mucins, sialyltransferases, and fucosyltransferases were analyzed in benign and malignant meningioma using the GEO database (GEO series GSE16581) and quantitative-PCR (qPCR). Results: Lectin cytochemistry revealed that the terminal galactose (Gal) and N-acetyl galactosamine (GalNAc) were highly expressed in primary benign meningioma cells (WHO grade I) compared to malignant meningioma cell lines (WHO grade III). The expression profile of mucin types O-glycosyltransferases in meningiomas were observed through the GEO database and gene expression experiment in meningioma cell lines. In the GEO database, C1GALT1-specific chaperone (COSMC) and mucin 1 (MUC1) were significantly increased in malignant meningiomas (Grade II and III) compared with benign meningiomas (Grade I). Meanwhile, in the cell lines, Core 2 ß1,6-N-acetylglucosaminyltransferase-2 (C2GNT2) was highly expressed in malignant meningiomas. We then investigated the complex mucin-type O-glycans structures by determination of sialyltransferases and fucosyltransferases. We found ST3 ß-galactoside α-2,3-sialyltransferase 4 (ST3GAL4) was significantly decreased in the GEO database, while ST3GAL1, ST3GAL3, α1,3 fucosyltransferases 1 and 8 (FUT1 and FUT8) were highly expressed in malignant meningioma cell lines-(HKBMM)-compared to primary benign meningioma cells-(SUT-MG12 and SUT-MG14). Conclusion: Our findings are the first to demonstrate the potential glycosylation changes in the O-linked glycans of malignant meningiomas compared with benign meningiomas, which may play an essential role in the progression, tumorigenesis, and malignancy of meningiomas.

Establishment and characterization of a novel cancer stem-like cell of cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive malignant tumor of bile duct epithelia. Recent evidence suggests the impact of cancer stem cells (CSC) on the therapeutic resistance of CCA; however, the knowledge of CSC in CCA is limited due to the lack of a CSC model. In this study, we successfully established a stable sphere-forming CCA stem-like cell, KKU-055-CSC, from the original CCA cell line, KKU-055. The KKU-055-CSC exhibits CSC characteristics, including: (1) the ability to grow stably and withstand continuous passage for a long period of culture in the stem cell medium, (2) high expression of stem cell markers, (3) low responsiveness to standard chemotherapy drugs, (4) multilineage differentiation, and (5) faster and constant expansive tumor formation in xenograft mouse models. To identify the CCA-CSC-associated pathway, we have undertaken a global proteomics and functional cluster/network analysis. Proteomics identified the 5925 proteins in total, and the significantly upregulated proteins in CSC compared with FCS-induced differentiated CSC and its parental cells were extracted. Network analysis revealed that high mobility group A1 (HMGA1) and Aurora A signaling through the signal transducer and activator of transcription 3 pathways were enriched in KKU-055-CSC. Knockdown of HMGA1 in KKU-055-CSC suppressed the expression of stem cell markers, induced the differentiation followed by cell proliferation, and enhanced sensitivity to chemotherapy drugs including Aurora A inhibitors. In silico analysis indicated that the expression of HMGA1 was correlated with Aurora A expressions and poor survival of CCA patients. In conclusion, we have established a unique CCA stem-like cell model and identified the HMGA1-Aurora A signaling as an important pathway for CSC-CCA.

SIRT7 suppresses energy expenditure and thermogenesis by regulating brown adipose tissue functions in mice.

Brown adipose tissue plays a central role in the regulation of the energy balance by expending energy to produce heat. NAD+-dependent deacylase sirtuins have widely been recognized as positive regulators of brown adipose tissue thermogenesis. However, here we reveal that SIRT7, one of seven mammalian sirtuins, suppresses energy expenditure and thermogenesis by regulating brown adipose tissue functions. Whole-body and brown adipose tissue-specific Sirt7 knockout mice have higher body temperature and energy expenditure. SIRT7 deficiency increases the protein level of UCP1, a key regulator of brown adipose tissue thermogenesis. Mechanistically, we found that SIRT7 deacetylates insulin-like growth factor 2 mRNA-binding protein 2, an RNA-binding protein that inhibits the translation of Ucp1 mRNA, thereby enhancing its inhibitory action on Ucp1. Furthermore, SIRT7 attenuates the expression of batokine genes, such as fibroblast growth factor 21. In conclusion, we propose that SIRT7 serves as an energy-saving factor by suppressing brown adipose tissue functions.

Emerging roles of GALNT5 on promoting EGFR activation in cholangiocarcinoma: a mechanistic insight.

Cholangiocarcinoma (CCA) is a lethal cancer in that the incidence is now increasing worldwide. N-acetylgalactosaminyltransferase 5 (GALNT5), an enzyme that initiates the first step of mucin type-O glycosylation, has been reported to promote aggressiveness of CCA cells via the epithelial to the mesenchymal transition (EMT) process, and Akt/Erk activation. In this study, the clinical and biological relevance of GALNT5 and the molecular mechanisms by which GALNT5 modulated EGFR in promoting CCA progression were examined. Using publicly available datasets, upregulation of GALNT5 in patient CCA tissues and its correlation with EGFR expression was noted. High levels of GALNT5 were significantly associated with the short survival of patients, suggesting a prognostic marker of GALNT5 for CCA. GALNT5 modulated EGFR expression as shown in CCA cell lines. Upregulation of GALNT5 significantly increased EGFR mRNA and protein in GALNT5 overexpressing cells, whereas suppression of GALNT5 expression gave the opposite results. The molecular dynamics simulations and MM/PB(GB)SA-based free energy calculations showed that O-glycosylation on the EGFR extracellular domain enhanced the structural stability, compactness, and H-bond formation of the EGF/GalNAc-EGFR complex compared with those of EGF/EGFR. This stabilized the growth factor binding site and fostered stronger interactions between EGF and EGFR. Using the EGF-induced EGFR activation model, GALNT5 was shown to mediate EGFR stability via a decreased rate of EGFR degradation and enhanced EGFR activity by increasing the binding affinity of EGF/EGFR that consequently increasing the activation of EGFR and its downstream effectors Akt and Erk. In summary, GALNT5 was upregulated in CCA tissues and associated with a worse prognosis. The study identified for the first time the impacts of GALNT5 on EGFR activity by increasing: 1) EGFR expression via a transcriptional-dependent mechanism, 2) EGFR stability by reducing EGFR degradation, and 3) EGFR activation through an increased binding affinity of EGF/EGFR which all together fostered the activation of EGFR. These results expanded the understanding of the molecular mechanism of how GALNT5 impacted CCA progression and suggested GALNT5 as a new target for therapeutic intervention against metastatic CCA.

AT-hook DNA-binding motif-containing protein one knockdown downregulates EWS-FLI1 transcriptional activity in Ewing's sarcoma cells.

Ewing's sarcoma is the second most common bone malignancy in children or young adults and is caused by an oncogenic transcription factor by a chromosomal translocation between the EWSR1 gene and the ETS transcription factor family. However, the transcriptional mechanism of EWS-ETS fusion proteins is still unclear. To identify the transcriptional complexes of EWS-ETS fusion transcription factors, we applied a proximal labeling system called BioID in Ewing's sarcoma cells. We identified AHDC1 as a proximal protein of EWS-ETS fusion proteins. AHDC1 knockdown showed a reduced cell growth and transcriptional activity of EWS-FLI1. AHDC1 knockdown also reduced BRD4 and BRG1 protein levels, both known as interacting proteins of EWS-FLI1. Our results suggest that AHDC1 supports cell growth through EWS-FLI1.

Water Droplet-in-Oil Digestion Method for Single-Cell Proteomics.

Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.

Targeting alpha2,3-sialylated glycan in glioma stem-like cells by Maackia amurensis lectin-II: A promising strategy for glioma treatment.

Glioma stem/initiating cells have been considered a major cause of tumor recurrence and therapeutic resistance. In this study, we have established a new glioma stem-like cell (GSC), named U373-GSC, from the U373 glioma cell line. The cells exhibited stemness properties, e.g., expression of stem cell markers, self-renewal activity, multi-lineage differentiating abilities, and drug resistance. Using U373-GSC and GSC-03A-a GSC clone previously established from patient tissue, we have identified a novel GSC-associated sialic acid-modified glycan commonly expressed in both cell lines. Lectin fluorescence staining showed that Maackia amurensis lectin II (MAL-II)-binding alpha2,3-sialylated glycan (MAL-SG) was highly expressed in GSCs, and drastically decreased during FBS induced differentiation to glioma cells or little in the parental cells. Treatment of GSCs by MAL-II, compared with other lectins, showed that MAL-II significantly suppresses cell viability and sphere formation via induction of cell cycle arrest and apoptosis of the GSCs. Similar effects were observed when the cells were treated with a sialyltransferase inhibitor or sialidase. Taken together, we demonstrate for the first time that MAL-SGs/alpha-2,3 sialylations are upregulated and control survival/maintenances of GSCs, and their functional inhibitions lead to apoptosis of GSCs. MAL-SG could be a potential marker and therapeutic target of GSCs; its inhibitors, such as MAL-II, may be useful for glioma treatment in the future.

Bioinformatic Prediction of Novel Signaling Pathways of Apoptosis-inducing Factor, Mitochondrion-associated 3 (AIFM3) and Their Roles in Metastasis of Cholangiocarcinoma Cells.

BACKGROUND/AIM: We previously demonstrated that a mitochondrial protein, apoptosis-inducing factor, mitochondrion-associated 3 (AIFM3) is over-expressed in cholangiocarcinoma (CCA) and its serum levels can be a prognostic biomarker for CCA. To elucidate the functional roles of AIFM3 in CCA progression, we aimed to determine the signaling pathways of AIFM3 in CCA. MATERIALS AND METHODS: AIFM3 gene in CCA cells was silenced and AIFM3-related proteins were identified using mass spectrometry and bioinformatics tools. The relationships between AIFM3 and 441 related proteins were explored. To validate the functions of AIFM3, transwell migration/invasion assays were used. RESULTS: Bioinformatic analyses predicted that AIFM3 interacts with formin-like protein 3 (FMNL3) and is involved in tumor cell motilities. Online database analysis revealed higher AIFM3 mRNA expression levels in CCA, particularly with lymph node metastasis. After AIFM3 gene silencing, CCA cell migration/invasion was significantly decreased (p<0.001). Furthermore, AIFM3 expression levels were significantly associated with lymph node metastasis (p=0.0009) and shorter survival time (p=0.020). CONCLUSION: The AIFM3 signaling pathway is mediated via FMNL3 and involved in metastasis, suggesting that AIFM3 might be a molecular target to prevent CCA metastasis.

EGFR-Targeted Pentacyclic Triterpene Analogues for Glioma Therapy.

Glioma, particularly its most malignant form, glioblastoma multiforme (GBM), is the most common and aggressive malignant central nervous system tumor. The drawbacks of the current chemotherapy for GBM have aroused curiosity in the search for targeted therapies. Aberrantly overexpressed epidermal growth factor receptor (EGFR) in GBM results in poor prognosis, low survival rates, poor responses to therapy and recurrence, and therefore EGFR-targeted therapy stands out as a promising approach for the treatment of gliomas. In this context, a series of pentacyclic triterpene analogues were subjected to in vitro and in silico assays, which were conducted to assess their potency as EGFR-targeted anti-glioma agents. In particular, compound 10 was the most potent anti-glioma agent with an IC50 value of 5.82 µM towards U251 human glioblastoma cells. Taking into account its low cytotoxicity to peripheral blood mononuclear cells (PBMCs), compound 10 exerts selective antitumor action towards Jurkat human leukemic T-cells. This compound also induced apoptosis and inhibited EGFR with an IC50 value of 9.43 µM compared to erlotinib (IC50 = 0.06 µM). Based on in vitro and in silico data, compound 10 stands out as a potential orally bioavailable EGFR-targeted anti-glioma agent endowed with the ability to cross the blood-brain barrier (BBB).

Purification, Characterization, and Gene Expression of Rice Endo-ß-N-Acetylglucosaminidase, Endo-Os.

In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-ß-N-acetylglucosaminidase (endo-ß-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-ß-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-ß-GlcNAc-ases and preliminarily reported the gene information of rice endo-ß-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-ß-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-ß-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Manα1-2Manα1-3Manß1 unit; this substrate specificity was almost the same as that of other plant endo-ß-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os.

Multi-serum glycobiomarkers improves the diagnosis and prognostic prediction of cholangiocarcinoma.

BACKGROUND: Aberrant glycosylation has been reported to play important roles in progression of cholangiocarcinoma (CCA) and hence the aberrant expressed glycans are beneficial markers for diagnosis and prognostic prediction of CCA. METHODS: Five CCA-associated glycobiomarkers-carbohydrate antigen 19-9 (CA19-9), carbohydrate antigen-S27 (CA-S27), CCA-associated carbohydrate antigen (CCA-CA), WFA-positive MUC1 (WFA+-MUC1), and WFA-positive M2BP (WFA+-M2BP), in the sera from CCA patients (N = 138) were determined in comparison with non-CCA control subjects (N = 246). RESULTS: Receiver operating characteristic analysis suggested the significance of each glycobiomarker in discriminating CCA from non-CCA with area under curve of 0.580-0.777. High levels of CA19-9, CCA-CA, CA-S27, or WFA+-MUC1 were associated with poor prognosis and poor survival of CCA patients. Combination of these glycobiomarkers and graded as a GlycoBiomarker (GB)-score could increase the power of the tests in diagnosis than an individual marker with 81% of sensitivity, specificity and accuracy. CONCLUSIONS: According to the GB-score, these glycobiomarkers not only increased diagnostic power but also discriminated survival of patients indicating the diagnostic and prognostic values of GB-score.

FTO Demethylates Cyclin D1 mRNA and Controls Cell-Cycle Progression.

N6-Methyladenosine (m6A) modification is the major chemical modification in mRNA that controls fundamental biological processes, including cell proliferation. Herein, we demonstrate that fat mass and obesity-associated (FTO) demethylates m6A modification of cyclin D1, the key regulator for G1 phase progression and controls cell proliferation in vitro and in vivo. FTO depletion upregulates cyclin D1 m6A modification, which in turn accelerates the degradation of cyclin D1 mRNA, leading to the impairment of G1 progression. m6A modification of cyclin D1 oscillates in a cell-cycle-dependent manner; m6A levels are suppressed during the G1 phase and enhanced during other phases. Low m6A levels during G1 are associated with the nuclear translocation of FTO from the cytosol. Furthermore, nucleocytoplasmic shuttling of FTO is regulated by casein kinase II-mediated phosphorylation of FTO. Our results highlight the role of m6A in regulating cyclin D1 mRNA stability and add another layer of complexity to cell-cycle regulation.

The O-GalNAcylating enzyme GALNT5 mediates carcinogenesis and progression of cholangiocarcinoma via activation of AKT/ERK signaling.

Mucin type O-glycosylation is a posttranslational modification of membrane and secretory proteins. Transferring of N-acetylgalactosamine, the first sugar of O-glycosylation, is catalyzed by one of the 20 isoforms of polypeptide N-acetylgalactosaminyltransferases (GALNTs). In this study, Vicia villosa lectin (VVL), a lectin that recognizes O-GalNAcylated glycans, was used to detect VVL-binding glycans (VBGs) in cholangiocarcinoma (CCA). The elevation of VBGs in tumor tissues of the liver fluke associated with CCA from hamsters and patients was noted. VBGs were detected in hyperplastic/dysplastic bile ducts and CCA but not in normal biliary epithelia and hepatocytes, indicating the association of VBGs with CCA development and progression. GALNT5 was shown to be the major isoform found in human CCA cell lines with high VBG expression. Suppression of GALNT5 expression using siRNA significantly reduced VBG expression, signifying the connection of GALNT5 and VBGs observed. Knocked-down GALNT5 expression considerably inhibited proliferation, migration and invasion of CCA cells. Increased expression of GALNT5 using pcDNA3.1-GALNT5 expression vector induced invasive phenotypes in CCA cells with low GALNT5 expression. Increasing of claudin-1 and decreasing of slug and vimentin expression together with inactivation of Akt/Erk signaling were noted in GALNT5 knocked-down cells. These observations were reversed in GALNT5 over-expressing cells. GALNT5-modulated progression of CCA cells was shown to be, in part, via GALNT5-mediated autocrine/paracrine factors that stimulated activations of Akt/Erk signaling and the epithelial to mesenchymal transition process. GALNT5 and its O-GalNAcylated products may have important roles in promoting progression of CCA and could possibly be novel targets for treatment of metastatic CCA.

Identification of a Specific Translational Machinery via TCTP-EF1A2 Interaction Regulating NF1-associated Tumor Growth by Affinity Purification and Data-independent Mass Spectrometry Acquisition (AP-DIA).

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease that predisposes individuals to developing benign neurofibromas and malignant peripheral nerve sheath tumors (MPNST). The mechanism of NF1-tumorigenesis or the curatives have not been established. Using unique trascriptome and proteome integration method, iPEACH (1), we previously identified translationally controlled tumor protein (TCTP) as a novel biological target for NF1-associated tumors (2). Here, we identified specific TCTP-interacting proteins by sequential affinity purification and data-independent mass spectrometry acquisition (AP-DIA/SWATH) to investigate the role of TCTP in NF1-associated malignant tumors. TCTP mainly interacts with proteins related to protein synthesis and especially to elongation factor complex components, including EF1A2, EF1B, EF1D, EF1G, and valyl-tRNA synthetase (VARS), in NF1-deficient malignant tumor cells. Interestingly, TCTP preferentially binds to EF1A2 (normally found only in neural and skeletal-muscle cells and several cancer cells), rather than EF1A1 despite the high homologies (98%) in their sequences. The docking simulation and further validations to study the interaction between TCTP and EF1A2 revealed that TCTP directly binds with EF1A2 via the contact areas of EF1A2 dimerization. Using unique and common sequences between EF1A2 and EF1A1 in AP-DIA/SWATH, we quantitatively validated the interaction of EF1A2 and TCTP/other elongation factors and found that TCTP coordinates the translational machinery of elongation factors via the association with EF1A2. These data suggest that TCTP activates EF1A2-dependent translation by mediating complex formation with other elongation factors. Inhibiting the TCTP-EF1A2 interaction with EF1A2 siRNAs or a TCTP inhibitor, artesunate, significantly down-regulated the factors related to protein translation and caused dramatic suppression of growth/translation in NF1-associated tumors. Our findings demonstrate that a specific protein translation machinery related to the TCTP-EF1A2 interaction is functionally implicated in the tumorigenesis and progression of NF1-associated tumors and could represent a therapeutic target.

The jPOST environment: an integrated proteomics data repository and database.

Rapid progress is being made in mass spectrometry (MS)-based proteomics, yielding an increasing number of larger datasets with higher quality and higher throughput. To integrate proteomics datasets generated from various projects and institutions, we launched a project named jPOST (Japan ProteOme STandard Repository/Database, https://jpostdb.org/) in 2015. Its proteomics data repository, jPOSTrepo, began operations in 2016 and has accepted more than 10 TB of MS-based proteomics datasets in the past two years. In addition, we have developed a new proteomics database named jPOSTdb in which the published raw datasets in jPOSTrepo are reanalyzed using standardized protocol. jPOSTdb provides viewers showing the frequency of detected post-translational modifications, the co-occurrence of phosphorylation sites on a peptide and peptide sharing among proteoforms. jPOSTdb also provides basic statistical analysis tools to compare proteomics datasets.

Isocitrate dehydrogenase gene mutations and 2-hydroxyglutarate accumulation in esophageal squamous cell carcinoma.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that convert isocitrate to α-ketoglutarate. Somatic point mutations in IDH1/2 confer a gain-of-function in cancer cells, resulting in overproduction of an oncometabolite, 2-hydroxyglutarate (2HG). 2HG interferes with cellular metabolism and epigenetic regulation, contributing to oncogenesis. Given that IDH1 and IDH2 are attracting attention as promising therapeutic targets, better evaluation of the incidence of IDH1 and IDH2 mutations and 2HG level in human cancers is clinically important. This is the first study to assess their incidence in esophageal squamous cell carcinomas (ESCCs). First, we established pyrosequencing assays for IDH1 and IDH2 mutations and revealed that these mutations were absent in 10 ESCC cell lines and 96 ESCC tissues. Second, utilizing IDH1 and IDH2 overexpression vectors, we demonstrated that LC-MS/MS assays can accurately evaluate 2HG level and found that some ESCC cases presented a high level of 2HG. In conclusion, IDH1 or IDH2 mutations play a limited role in the development of ESCC. 2HG is potentially synthesized to high levels in the absence of IDH1 and IDH2 mutations, and this may correlate with progression of ESCCs.

Artesunate and chloroquine induce cytotoxic activity on cholangiocarcinoma cells via different cell death mechanisms.

Chemotherapy for cholangiocarcinoma (CCA) is not quite successful. In this study, we revisited the possibility of artesunate (ART) and chloroquine (CQ), the antimalarial drugs, as therapeutic agents against CCA. The possible mechanisms of these drugs to exert cytotoxicity on CCA cells were also explored. The effects of ART and CQ on proliferation and death patterns of two CCA cell lines, KKU-214 and its highly metastatic subtype KKU-214L5, were examined using water soluble tetrazolium (WST) assay and time-lapse photometry, respectively. To differentiate and verify the death patterns between necrosis and apoptosis, lactate dehydrogenase (LDH) release, and caspase 3 activity were measured. CellROXTM green reagent staining method was used to assess reactive oxygen species (ROS) production in ART- and CQ-treated cells. ART and CQ significantly inhibited proliferation of CCA cells. Both drugs kill malarial parasites via similar mechanism depending on ROS formation, however, ART induced necrotic cell death and CQ induced apoptotic cell death in CCA cells. ART induced LDH release, whereas CQ activated caspase 3, confirming induction of necrotic and apoptotic cell deaths by ART and CQ, respectively. ART treatment induced higher ROS production than CQ. ART and CQ induce CCA cells death via different death pathways. ART should be suitable for necrosis-sensitive CCA, whereas CQ is more suitable for apoptosis-sensitive CCA.

SIRT7 has a critical role in bone formation by regulating lysine acylation of SP7/Osterix.

SP7/Osterix (OSX) is a master regulatory transcription factor that activates a variety of genes during differentiation of osteoblasts. However, the influence of post-translational modifications on the regulation of its transactivation activity is largely unknown. Here, we report that sirtuins, which are NAD(+)-dependent deacylases, regulate lysine deacylation-mediated transactivation of OSX. Germline Sirt7 knockout mice develop severe osteopenia characterized by decreased bone formation and an increase of osteoclasts. Similarly, osteoblast-specific Sirt7 knockout mice showed attenuated bone formation. Interaction of SIRT7 with OSX leads to the activation of transactivation by OSX without altering its protein expression. Deacylation of lysine (K) 368 in the C-terminal region of OSX by SIRT7 promote its N-terminal transactivation activity. In addition, SIRT7-mediated deacylation of K368 also facilitates depropionylation of OSX by SIRT1, thereby increasing OSX transactivation activity. In conclusion, our findings suggest that SIRT7 has a critical role in bone formation by regulating acylation of OSX.

RBMX is a component of the centromere noncoding RNP complex involved in cohesion regulation.

Satellite I RNA, a noncoding (nc)RNA transcribed from repetitive regions in human centromeres, binds to Aurora kinase B and forms a ncRNP complex required for chromosome segregation. To examine its function in this process, we purified satellite I ncRNP complex from nuclear extracts prepared from asynchronized or mitotic (M) phase-arrested HeLa cells and then carried out LC/MS to identify proteins bound to satellite I RNA. RBMX (RNA-binding motif protein, X-linked), which was isolated from M phase-arrested cells, was selected for further characterization. We found that RBMX associates with satellite I RNA only during M phase. Knockdown of RBMX induced premature separation of sister chromatid cohesion and abnormal nuclear division. Likewise, knockdown of satellite I RNA also caused premature separation of sister chromatids during M phase. The amounts of RBMX and Sororin, a cohesion regulator, were reduced in satellite I RNA-depleted cells. These results suggest that satellite I RNA plays a role in stabilizing RBMX and Sororin in the ncRNP complex to maintain proper sister chromatid cohesion.