Resistance to DNA-damaging agents is a significant cause of treatment failure and poor outcomes in oncology. To identify unrecognized regulators of cell survival we performed a whole-genome ...CRISPR-Cas9 screen using treatment with ionizing radiation as a selective pressure, and identified STING (stimulator of interferon genes) as an intrinsic regulator of tumor cell survival. We show that STING regulates a transcriptional program that controls the generation of reactive oxygen species (ROS), and that STING loss alters ROS homeostasis to reduce DNA damage and to cause therapeutic resistance. In agreement with these data, analysis of tumors from head and neck squamous cell carcinoma patient specimens show that low STING expression is associated with worse outcomes. We also demonstrate that pharmacologic activation of STING enhances the effects of ionizing radiation in vivo, providing a rationale for therapeutic combinations of STING agonists and DNA-damaging agents. These results highlight a role for STING that is beyond its canonical function in cyclic dinucleotide and DNA damage sensing, and identify STING as a regulator of cellular ROS homeostasis and tumor cell susceptibility to reactive oxygen dependent, DNA damaging agents.
Three cholangiocarcinoma (CCA) cell line—formerly named, M156, M213 and M214 have been intensively used with discrepancy of their tumor origins. They were assumed to be originated from three ...different donors without authentication. To verify the origins of these cell lines, the short tandem repeat (STR) analysis of the currently used cell lines, the cell stocks from the establisher and the primary tumor of a CCA patient were performed. Their phenotypic and genotypic originality were compared. The currently used 3 CCA cell lines exhibited similar STR as CCA patient ID-M213 indicating the same origin of these cells. The cell stocks from the establisher, however, revealed the same STR of M213 and M214 cells, but not M156. The misidentification of M214 and M156 is probably due to the mislabeling and cross-contamination of M213 cells during culture. These currently used cell lines were renamed as KKU-213A, -213B and -213C, for the formerly M213, M214 and M156 cells, respectively. These cell lines were established from a male with an intrahepatic mass-forming CCA stage-4B. The tumor was an adenosquamous carcinoma with the liver fluke ova granuloma in evidence. All cell lines had positive CK19 with differential CA19-9 expression. They exhibited aneuploidy karyotypes, distinct cell morphology, cell growth, cytogenetic characteristic and progressive phenotypes. KKU-213C formed a adenosquamous carcinoma, whereas KKU-213A and KKU-213B formed poorly- and well-differentiated squamous cell carcinomas in xenografted mice. mRNA microarray revealed different expression profiles among these three cell lines. The three cell lines have unique characteristics and may resemble the heterogeneity of tumor origin.
Increased glucose utilization is a feature of cancer cells to support cell survival, proliferation, and metastasis. An association between diabetes mellitus and cancer progression was previously ...demonstrated in cancers including cholangiocarcinoma (CCA). This study was aimed to determine the effects of high glucose on protein O-GlcNAcylation and metastatic potentials of CCA cells. Two pairs each of the parental low metastatic and highly metastatic CCA sublines were cultured in normal (5.6 mM) or high (25 mM) glucose media. The migration and invasion abilities were determined and underlying mechanisms were explored. Results revealed that high glucose promoted migration and invasion of CCA cells that were more pronounced in the highly metastatic sublines. Concomitantly, high glucose increased global O-GlcNAcylated proteins, the expressions of vimentin, hexokinase, glucosamine-fructose-6-phosphate amidotransferase (GFAT) and O-GlcNAc transferase of CCA cells. The glucose level that promoted migration/invasion was shown to be potentiated by the induction of GFAT, O-GlcNAcylation and an increase of O-GlcNAcylated vimentin and vimentin expression. Treatment with a GFAT inhibitor reduced global O-GlcNAcylated proteins, vimentin expression, and alleviated cell migration. Altogether, these results suggested the role of high glucose enhanced CCA metastasis via modulation of O-GlcNAcylation, through the expressions of GFAT and vimentin.
Membrane-bound oligosaccharides form the interfacial boundary between the cell and its environment, mediating processes such as adhesion and signaling. These structures can undergo dynamic changes in ...composition and expression based on cell type, external stimuli, and genetic factors. Glycosylation, therefore, is a promising target of therapeutic interventions for presently incurable forms of advanced cancer. Here, we show that cholangiocarcinoma metastasis is characterized by down-regulation of the Golgi α-mannosidase I coding gene MAN1A1, leading to elevation of extended highmannose glycans with terminating α-1,2-mannose residues. Subsequent reshaping of the glycome by inhibiting α-mannosidase I resulted in significantly higher migratory and invasive capabilities while masking cell surface mannosylation suppressed metastasisrelated phenotypes. Exclusive elucidation of differentially expressed membrane glycoproteins and molecular modeling suggested that extended high-mannose glycosylation at the helical domain of transferrin receptor protein 1 promotes conformational changes that improve noncovalent interaction energies and lead to enhancement of cell migration in metastatic cholangiocarcinoma. The results provide support that α-1,2-mannosylated N-glycans present on cancer cell membrane proteins may serve as therapeutic targets for preventing metastasis.
Berberine is a natural compound found in several herbs. Anticancer activity of berberine was reported in several cancers, however, little is known regarding the effects of berberine against ...cholangiocarcinoma (CCA). In this study, the growth inhibitory effects of berberine on CCA cell lines and its molecular mechanisms were explored. Cell growth and cell cycle distribution were examined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. The expression levels of cell cycle regulatory proteins were determined by Western blot analysis. Berberine significantly inhibited growth of CCA cell lines in a dose and time dependent fashion. The inhibition was largely attributed to cell cycle arrest at the G1 phase through the reduction of cyclin D1, and cyclin E. Moreover, berberine could reduce the expression and activation of signal transducers and activator of transcription 3 (STAT3) and probably nuclear factor-kappaB (NF-κB) via suppression of extracellular signal-regulated kinase (ERK) 1/2 action. These results highlight the potential of berberine to be a multi-target agent for CCA treatment.
Epidemiological studies indicate diabetes mellitus and hyperglycemia as risk factors of cancers including cholangiocarcinoma (CCA). How high glucose promotes cancer development and progression, ...however, is still unrevealed. In this study, insight into the molecular pathway of high glucose promoting progression of CCA cells was investigated.
Human CCA cell lines, KKU-213A and KKU-213B were cultured in normal glucose (NG; 5.56 mM) or high glucose (HG; 25 mM) and used as NG and HG cells. Forkhead box M1 (FOXM1) expression was transiently suppressed using siFOXM1. Western blotting and image analysis were employed to semi-quantitatively determine the expression levels of the specified proteins. The migration and invasion of CCA cells were revealed using Boyden chamber assays.
All HG cells exhibited higher expression of FOXM1 than the corresponding NG cells in a dose dependent manner. Suppression of FOXM1 expression by siFOXM1 significantly reduced migration and invasion abilities of CCA cells by suppression of Slug and MMP2 expression. Inhibition of STAT3 activation using Stattic, significantly suppressed expression of FOXM1 and Slug and decreased migration and invasion abilities of HG cells. In addition, EGFR expression was significantly higher in HG cells than NG cells and increased dependently with glucose concentration. Inhibition of EGFR activation by cetuximab significantly suppressed STAT3 activation and FOXM1 expression.
The mechanism of high glucose promoting progression of CCA cells was revealed to be via in part by upregulation of FOXM1 expression under EGF/EGFR and STAT3 dependent activation.
•FOXM1 expression in CCA cells was increased according to glucose concentrations.•FOXM1 promotes migration and invasion of CCA cells.•Suppression of STAT3 activation reduced FOXM1 expression and CCA progression.•High glucose promotes CCA progression via FOXM1, EGF/EGFR and STAT3 activation.
Aberrant glycosylation is recognized as a cancer hallmark that is associated with cancer development and progression. In this study, the clinical relevance and significance of terminal fucose (TFG), ...by fucosyltransferase-1 (FUT1) in carcinogenesis and progression of cholangiocarcinoma (CCA) were demonstrated. TFG expression in human and hamster CCA tissues were determined using Ulex europaeus agglutinin-I (UEA-I) histochemistry. Normal bile ducts rarely expressed TFG while 47% of CCA human tissues had high TFG expression and was correlated with shorter survival of patients. In the CCA-hamster model, TFG was elevated in hyperproliferative bile ducts and gradually increased until CCA was developed. This evidence indicates the involvement of TFG in carcinogenesis and progression of CCA. The mechanistic insight was performed in 2 CCA cell lines. Suppression of TFG expression using siFUT1 or neutralizing the surface TFG with UEA-I significantly reduced migration, invasion and adhesion of CCA cells in correlation with the reduction of Akt/Erk signaling and epithelial-mesenchymal transition. A short pulse of EGF could stimulate Akt/Erk signaling via activation of EGF-EGFR cascade, however, decreasing TFG using siFUT1 or UEA-I treatment reduced the EGF-EGFR activation and Akt/Erk signaling. This evidence provides important insight into the relevant role and molecular mechanism of TFG in progression of CCA.
O‐GlcNAcylation is a key post‐translational modification that modifies the functions of proteins. Associations between O‐GlcNAcylation, shorter survival of cholangiocarcinoma (CCA) patients, and ...increased migration/invasion of CCA cell lines have been reported. However, the specific O‐GlcNAcylated proteins (OGPs) that participate in promotion of CCA progression are poorly understood. OGPs were isolated from human CCA cell lines, KKU‐213 and KKU‐214, using a click chemistry‐based enzymatic labeling system, identified using LC‐MS/MS, and searched against an OGP database. From the proteomic analysis, a total of 21 OGPs related to cancer progression were identified, of which 12 have not been previously reported. Among these, hnRNP‐K, a multifaceted RNA‐ and DNA‐binding protein known as a pre‐mRNA‐binding protein, was one of the most abundantly expressed, suggesting its involvement in CCA progression. O‐GlcNAcylation of hnRNP‐K was further verified by anti‐OGP/anti‐hnRNP‐K immunoprecipitations and sWGA pull‐down assays. The perpetuation of CCA by hnRNP‐K was evaluated using siRNA, which revealed modulation of cyclin D1, XIAP, EMT markers, and MMP2 and MMP7 expression. In native CCA cells, hnRNP‐K was primarily localized in the nucleus; however, when O‐GlcNAcylation was suppressed, hnRNP‐K was retained in the cytoplasm. These data signify an association between nuclear accumulation of hnRNP‐K and the migratory capabilities of CCA cells. In human CCA tissues, expression of nuclear hnRNP‐K was positively correlated with high O‐GlcNAcylation levels, metastatic stage, and shorter survival of CCA patients. This study demonstrates the significance of O‐GlcNAcylation on the nuclear translocation of hnRNP‐K and its impact on the progression of CCA.
Elevation of O‐GlcNAcylation is associated with progression of cholangiocarcinoma (CCA). hnRNP‐K, a pre‐mRNA‐binding protein, was identified as one of the most abundant O‐GlcNAcylated proteins in CCA cell lines. O‐GlcNAcylation correlates with nuclear translocation of hnRNP‐K and progression of CCA cells via effector molecules related to proliferation, anti‐apoptosis, migration and invasion.
O-GlcNAcylation, an O-linked protein glycosylation with a single molecule of N-acetylglucosamine (GlcNAc), is reversibly controlled by O-GlcNAc transferase (OGT) and N-acetyl D-glucosaminidase (OGA). ...Aberrant O-GlcNAcylation contributes an important role in initiation and progression of many human cancers. Elevation of O-GlcNAcylation in tumor tissues and poor prognosis of cholangiocarcinoma (CCA) patients have been reported. In this study, the role of O-GlcNAcylation in promoting tumor progression was further investigated in CCA cell lines. Suppression of O-GlcNAcylation using small interfering RNAs of OGT (siOGT) significantly reduced cell migration and invasion of CCA cells whereas siOGA treated cells exhibited opposite effects. Manipulating levels of O-GlcNAcylation did affect the nuclear translocation of NF-κB and Akt-phosphorylation together with expression of matrix-metalloproteinases (MMPs). O-GlcNAcylation and nuclear translocation of NF-κB, the upstream signaling cascade of MMP activation were shown to be important for MMP activation. Immunoprecipitation revealed the elevation of O-GlcNAc-modified NF-κB with increased cellular O-GlcNAcylation. Involvement of O-GlcNAcylation in MMP-mediated migration and invasion of CCA cells was shown to be via O-GlcNAcylation and nuclear translocation of NF-κB. This information indicates the significance of O-GlcNAcylation in controlling the metastatic ability of CCA cells, hence, O-GlcNAcylation and its products may be new targets for treatment of metastatic CCA.
Secreted proteins are overexpressed in cholangiocarcinoma (CCA) and actively involved in promoting metastatic spread. Many of these proteins possess one or more sites of glycosylation and their ...various glycoforms have potential utility as prognostic or diagnostic biomarkers. To evaluate the effects of secretome glycosylation on patient outcome, we elucidated the glycosylation patterns of proteins secreted by parental and metastatic CCA cells using liquid chromatography‐mass spectrometry. Our analysis showed that the secretome of CCA cells was dominated by fucosylated and fucosialylated glycoforms. Based on the glycan and protein profiles, we evaluated the combined prognostic significance of glycosyltransferases and secretory proteins. Significantly, genes encoding fucosyltransferases and sialyltransferases showed favorable prognostic effects when combined with secretory protein‐coding gene expression, particularly thrombospondin‐1. Combining these measures may provide improved risk assessment for CCA and be used to indicate stages of disease progression.
Changes in fucosylation and sialylation were associated with the secretome of metastatic cholangiocarcinoma (CCA). A survival analysis across secreted protein‐coding genes revealed a more favorable prognostic effect when combined with fucosyltransferase‐ or sialyltransferase‐coding genes than when evaluated individually. Glycosylation enzymes improved the prognostic value of secreted proteins in CCA.
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