Exosomal HMGA2 protein from EBV-positive NPC cells destroys vascular endothelial barriers and induces endothelial-to-mesenchymal transition to promote metastasis.

Increased vascular permeability facilitates metastasis. Cancer-secreted exosomes are emerging mediators of cancer-host crosstalk. Epstein-Barr virus (EBV), identified as the first human tumor-associated virus, plays a crucial role in metastatic tumors, especially in nasopharyngeal carcinoma (NPC). To date, whether and how exosomes from EBV-infected NPC cells affect vascular permeability remains unclear. Here, we show that exosomes from EBV-positive NPC cells, but not exosomes from EBV-negative NPC cells, destroy endothelial cell tight junction (TJ) proteins, which are natural barriers against metastasis, and promote endothelial-to-mesenchymal transition (EndMT) in endothelial cells. Proteomic analysis revealed that the level of HMGA2 protein was higher in exosomes derived from EBV-positive NPC cells compared with that in exosomes derived from EBV-negative NPC cells. Depletion of HMGA2 in exosomes derived from EBV-positive NPC cells attenuates endothelial cell dysfunction and tumor cell metastasis. In contrast, exosomes from HMGA2 overexpressing EBV-negative NPC cells promoted these processes. Furthermore, we showed that HMGA2 upregulates the expression of Snail, which contributes to TJ proteins reduction and EndMT in endothelial cells. Moreover, the level of HMGA2 in circulating exosomes is significantly higher in NPC patients with metastasis than in those without metastasis and healthy negative controls, and the level of HMGA2 in tumor cells is associated with TJ and EndMT protein expression in endothelial cells. Collectively, our findings suggest exosomal HMGA2 from EBV-positive NPC cells promotes tumor metastasis by targeting multiple endothelial TJ and promoting EndMT, which highlights secreted HMGA2 as a potential therapeutic target and a predictive marker for NPC metastasis.

Epstein-Barr Virus Induces Lymphangiogenesis and Lympth Node Metastasis via Upregulation of VEGF-C in Nasopharyngeal Carcinoma.

Lymphatic metastasis is a common clinical symptom in nasopharyngeal carcinoma (NPC), the most common Epstein-Barr virus (EBV)-associated head and neck malignancy. However, the effect of EBV on NPC lymph node (LN) metastasis is still unclear. In this study, we demonstrated that EBV infection is strongly associated with advanced clinical N stage and lymphangiogenesis of NPC. We found that NPC cells infected with EBV promote LN metastasis by inducing cancer-associated lymphangiogenesis, whereas these changes were abolished upon clearance of EBV genomes. Mechanistically, EBV-induced VEGF-C contributed to lymphangiogenesis and LN metastasis, and PHLPP1, a target of miR-BART15, partially contributed to AKT/HIF1a hyperactivity and subsequent VEGF-C transcriptional activation. In addition, administration of anti-VEGF-C antibody or HIF1α inhibitors attenuated the lymphangiogenesis and LN metastasis induced by EBV. Finally, we verified the clinical significance of this prometastatic EBV/VEGF-C axis by determining the expression of PHLPP1, AKT, HIF1a, and VEGF-C in NPC specimens with and without EBV. These results uncover a reasonable mechanism for the EBV-modulated LN metastasis microenvironment in NPC, indicating that EBV is a potential therapeutic target for NPC with lymphatic metastasis. IMPLICATIONS: This research demonstrates that EBV induces lymphangiogenesis in NPC by regulating PHLPP1/p-AKT/HIF1a/VEGF-C, providing a new therapeutic target for NPC with lymphatic metastasis.

Discovery of a Series of 1,2,3-Triazole-Containing Erlotinib Derivatives With Potent Anti-Tumor Activities Against Non-Small Cell Lung Cancer.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are emerging at the vanguard of therapy for non-small-cell lung cancer (NSCLC) patients with EGFR-activating mutations. However, the increasing therapeutic resistance caused by novel mutations or activated bypass pathways has impaired their performance. In this study, we link one of the commercial EGFR-TKIs, Erlotinib, to different azide compounds to synthesize a novel class of 1,2,3-triazole ring-containing Erlotinib derivatives. We discovered that several new compounds show robust antiproliferation activity against diverse NSCLC cells in vitro including PC-9, H460, H1975 and A549. Two of the most potent compounds, e4 and e12 have been found to be more efficient than Erlotinib in all NSCLC cell lines except PC-9. They significantly induce apoptosis and cell cycle arrest in PC-9 and H460 cells. The antitumor efficacy of compound e4 in vivo is close to that of Erlotinib in a PC-9 xenograft mouse model. Most Erlotinib-1,2,3-triazole compounds exhibit moderate to good inhibitory activities toward wild-type EGFR as indicated by enzyme-linked immunosorbent assay (ELISA), and the EGFR phosphorylation was inhibited in H460 and PC-9 cells exposed to e4 or e12. These data suggest that these Erlotinib-1,2,3-triazole compounds are suitable candidates for use against NSCLC and more unknown mechanisms merit further investigation.

Synthesis and Antitumor Activity of Erlotinib Derivatives Linked With 1,2,3-Triazole.

Cervical cancer is one of the most important cause of cancer-related death and presents a major public health problem in many countries. To search for more novel antitumor agents against cervical cancer, 14 erlotinib-linked 1,2,3-triazole compounds were designed, synthesized, and evaluated for their anti-tumor activity. The compounds were confirmed by 1H NMR, 13C NMR, and high-resolution mass spectra (HR MS). Antitumor activity assay results indicated that six of those compounds have remarkable inhibitory activity against human cervical cancer HeLa cells in vitro, among which compound 4m was the most potent with IC50 of 3.79 µM, and compounds 4k, 4i, 4l, 4d, and 4n also demonstrated remarkable antitumor activity with IC50 of 3.79, 4.16, 4.36, 7.02, and 8.21 µM. We found three of the most potent compounds 4d, 4k, and 4l induced potent apoptosis and cell cycle arrest in HeLa cells, and compounds 4d and 4l significantly restrained the cell colony formation and showed moderate epidermal growth factor receptor (EGFR) inhibitory activity with IC50 of 13.01 and 1.76 µM. Therefore, these experiments indicate that these erlotinib-linked 1,2,3-triazole compounds are potential to act as effective anticancer agents against cervical cancer.

Author Correction: Epstein-Barr virus-encoded microRNA BART1 induces tumour metastasis by regulating PTEN-dependent pathways in nasopharyngeal carcinoma.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

A novel miR-200c/c-myc negative regulatory feedback loop is essential to the EMT process, CSC biology and drug sensitivity in nasopharyngeal cancer.

Overexpression of the c-Myc oncogene has been implicated in cancer stem cell - like (CSC) phenotypes and epithelial-to-mesenchymal transition (EMT) in cancer. However, the underlying molecular mechanism by which c-Myc regulates EMT and CSC potential in remains unclear. In the present study, we showed that the expression of c-Myc protein is inversely correlated with microRNA (miR)-200c expression in primary tumor samples from nasopharyngeal cancer (NPC) patients. We further demonstrated that Myc and miR-200c negatively regulate the expression each other in NPC cell lines. c-Myc transcriptionally repressed expression of miR-200c by directly binding to two E-box sites located within a 1 kb segment upstream of TSS of the miR-200c. In addition, miR-200c post-transcriptionally repressed expression of c-Myc by binding to its 3'-untranslated region, suggesting the existence of a negative feedback loop between Myc and miR-200c. Overexpression of c-Myc interfered with this feedback loop and activated the EMT program, induced CSC phenotypes, and enhanced drug sensitivity, whereas miR-200c could counteract these biological effects of c-Myc. Our results provide a novel mechanism governing c-Myc and miR-200c expression and indicate that either targeting c-Myc or restoring miR-200c expression would be a promising approach to overcome oncogenic role of c-Myc in NPC.

GenCLiP 3: mining human genes' functions and regulatory networks from PubMed based on co-occurrences and natural language processing.

SUMMARY: We present a web server, GenCLiP 3, which is an updated version of GenCLiP 2.0 to enhance analysis of human gene functions and regulatory networks, with the following improvements: i) accurate recognition of molecular interactions with polarity and directionality from the entire PubMed database; ii) support for Boolean search to customize multiple-term search and to quickly retrieve function related genes; iii) strengthened association between gene and keyword by a new scoring method; and iv) daily updates following literature release at PubMed FTP. AVAILABILITY: The server is freely available for academic use at: http://ci.smu.edu.cn/genclip3/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

CuS-MnS2 nano-flowers for magnetic resonance imaging guided photothermal/photodynamic therapy of ovarian cancer through necroptosis.

With a high incidence and high mortality rate, ovarian cancer presents a challenge for clinical practice. It is thus extremely urgent to investigate new diagnosis and therapy methods for the treatment of ovarian cancer. Ternary copper-based chalcogenide nanomaterials are attractive owing to their near infrared (NIR) response for cancer theranostic fields. However, improving the theranostic efficiency of these nanomaterials is challenging. Herein, CuS-MnS2 nano-flowers were easily synthesized and under NIR irradiation exhibited a relatively high photothermal conversion efficiency of 67.5% and a simultaneous reactive oxygen species (ROS) generation effect. Owing to these outstanding photothermal/photodynamic effects, excellent tumor ablation results could be achieved by the combined use of CuS-MnS2 nano-flowers and 808 nm NIR laser treatments. The main anticancer mechanism of CuS-MnS2 nano-flowers + NIR was likely necroptosis. In addition, the nano-flowers showed remarkable contrast enhancement according to magnetic resonance imaging (MRI). These CuS-MnS2 nano-flowers could thus serve as a promising multifunctional nanotheranostic agent for MRI and as a photothermal/photodynamic cancer therapy agent through necroptosis.

Core pluripotency factors promote glycolysis of human embryonic stem cells by activating GLUT1 enhancer.

Human embryonic stem cells (hESCs) depend on glycolysis for energy and substrates for biosynthesis. To understand the mechanisms governing the metabolism of hESCs, we investigated the transcriptional regulation of glucose transporter 1 (GLUT1, SLC2A1), a key glycolytic gene to maintain pluripotency. By combining the genome-wide data of binding sites of the core pluripotency factors (SOX2, OCT4, NANOG, denoted SON), chromosomal interaction and histone modification in hESCs, we identified a potential enhancer of the GLUT1 gene in hESCs, denoted GLUT1 enhancer (GE) element. GE interacts with the promoter of GLUT1, and the deletion of GE significantly reduces the expression of GLUT1, glucose uptake and glycolysis of hESCs, confirming that GE is an enhancer of GLUT1 in hESCs. In addition, the mutation of SON binding motifs within GE reduced the expression of GLUT1 as well as the interaction between GE and GLUT1 promoter, indicating that the binding of SON to GE is important for its activity. Therefore, SON promotes glucose uptake and glycolysis in hESCs by inducing GLUT1 expression through directly activating the enhancer of GLUT1.

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.

A miRNA-HERC4 pathway promotes breast tumorigenesis by inactivating tumor suppressor LATS1.

The E3 ligase HERC4 is overexpressed in human breast cancer and its expression levels correlated with the prognosis of breast cancer patients. However, the roles of HERC4 in mammary tumorigenesis remain unclear. Here we demonstrate that the knockdown of HERC4 in human breast cancer cells dramatically suppressed their proliferation, survival, migration, and tumor growth in vivo, while the overexpression of HERC4 promoted their aggressive tumorigenic activities. HERC4 is a new E3 ligase for the tumor suppressor LATS1 and destabilizes LATS1 by promoting the ubiquitination of LATS1. miRNA-136-5p and miRNA-1285-5p, expression of which is decreased in human breast cancers and is inversely correlated with the prognosis of breast cancer patients, are directly involved in suppressing the expression of HERC4. In summary, we discover a miRNA-HERC4-LATS1 pathway that plays important roles in the pathogenesis of breast cancer and represents new therapeutic targets for human breast cancer.

Casticin inhibits nasopharyngeal carcinoma growth by targeting phosphoinositide 3-kinase.

BACKGROUND: Casticin, an isoflavone compound extracted from the herb Fructus Viticis, has demonstrated anti-inflammatory and anticancer activities and properties. The aim of this study was to investigate the effects and mechanisms of casticin in nasopharyngeal carcinoma (NPC) cells and to determine its potential for targeted use as a medicine. METHODS: NPC cells were used to perform the experiments. The CCK­8 assay and colony formation assays were used to assess cell viability. Flow cytometry was used to measure the cell cycle and apoptosis analysis (annexin V/PI assay). A three-dimensional (3D) tumour sphere culture system was used to characterize the effect of casticin on NPC stem cells. In silico molecular docking prediction and high-throughput KINOME scan assays were used to evaluate the binding of casticin to phosphoinositide 3-kinase (PI3K), including wild-type and most of mutants variants. We also used the SelectScreen assay to detect the IC50 of ATP activity in the active site of the target kinase. Western blotting was used to evaluate the changes in key proteins involved cell cycle, apoptosis, stemness, and PI3K/protein kinase B (AKT) signalling. The effect of casticin treatment in vivo was determined by using a xenograft mouse model. RESULTS: Our results indicate that casticin is a new and novel selective PI3K inhibitor that can significantly inhibit NPC proliferation and that it induces G2/GM arrest and apoptosis by upregulating Bax/BCL2 expression. Moreover, casticin was observed to affect the self-renewal ability of the nasopharyngeal carcinoma cell lines, and a combination of casticin with BYL719 was observed to induce a decrease in the level of the phosphorylation of mTORC1 downstream targets in BYL719-insensitive NPC cell lines. CONCLUSION: Casticin is a newly emerging selective PI3K inhibitor with potential for use as a targeted therapeutic treatment for nasopharyngeal carcinoma. Accordingly, casticin might represent a novel and effective agent against NPC and likely has high potential for combined use with pharmacological agents targeting PI3K/AKT.

Piperidine nitroxide Tempol enhances cisplatin-induced apoptosis in ovarian cancer cells.

A nitroxide radical, Tempol (Tempol, TPL), is usually used as an antioxidative agent clinically, whereas the mechanism underlying its pro-oxidative effect has not been thoroughly investigated. The present study investigated the pro-oxidative effect of TPL on the inhibition of cellular proliferation and its role in enhancing the effect of anticancer drug cisplatin (DDP) on the induction of apoptosis in ovarian cancer cells. Cell viability and proliferation were evaluated by MTT assay. Cell apoptosis was analyzed by flow cytometry (FCM) following staining with Annexin V/propidium iodide. Western blot analysis was performed to determine the expression levels of anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and pro-apoptotic protein Bcl-2-associated X protein (Bax), and the Bcl-2:Bax expression ratio. Cellular reactive oxygen species (ROS) were labeled with dichlorofluorescin-diacetate and analyzed by FCM. The results revealed that cell viabilities of OVCAR3 and SKOV3 cells were decreased by TPL in dose-dependent manner at concentrations of 2 to 10 mM after 48 h incubation. The cell proliferation rates of OVCAR3 and SKOV3 cells were suppressed by TPL at lower toxic concentrations of 1.5 and 1 mM, respectively, compared with the control group. The MTT assay indicated that the combination therapy significantly inhibited the cell proliferation of OVCAR3 cells compared with treatment with DDP alone. FCM demonstrated that the combination treatment increased the proportion of early apoptotic cells in OVCAR3 cells compared with single DDP treatment. Western blot analysis revealed that the combination treatment markedly decreased the Bcl-2:Bax expression ratio compared with treatment with DDP alone. Detection of cellular ROS expression levels demonstrated that the combination therapy significantly increased cellular ROS generation compared with the DDP-only therapy. These data indicated that TPL increased the effect of DDP on inducing apoptosis in OVCAR3 cells.

[Generation of a new strain of NOD/SCID/IL2Rγ-/- mice with targeted disruption of Prkdc and IL2Rγ genes using CRISPR/Cas9 system].

OBJECTIVE: The NOD/SCID/IL2Rγ- /- (NSG) mouse strain is the most widely used immunodeficient strain for xenograft transplantation. However, the existing SCID mutation is a spontaneous mutation of the Prkdc gene, which leads to leaky T cell developmental block and difficulty in genotyping. It is therefore important to develop a new strain of NSG mice with targeted disruption of Prkdc and IL2Rγ genes. METHODS: Targeted disruption of Prkdc and IL2Rγ genes was achieved using the CRISPR/Cas9 system. By intercrossing the knockout and NOD mice, we obtained a novel strain of NOD/SCID/IL2Rγ- /-(NSG) mice, denoted as cNSG (Chinese NSG) mice. RESULTS: In addition to the NOD mutation, cNSG mice exhibited a complete absence of T cells, B cells and NK cells. cNSG mice allowed more efficient engraftment of human cancer cells than the commonly used immunodeficient nude mice. CONCLUSION: cNSG mice will provide an important xenotransplantation model for biomedical research.

A regulatory mutant on TRIM26 conferring the risk of nasopharyngeal carcinoma by inducing low immune response.

The major histocompatibility complex (MHC) is most closely associated with nasopharyngeal carcinoma (NPC), but the complexity of its genome structure has proven challenging for the discovery of causal MHC loci or genes. We conducted a targeted MHC sequencing in 40 Cantonese NPC patients followed by a two-stage replication in 1065 NPC cases and 2137 controls of Southern Chinese descendent. Quantitative RT-PCR analysis (qRT-PCR) was used to detect gene expression status in 108 NPC and 43 noncancerous nasopharyngeal (NP) samples. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) were used to assess the transcription factor binding site. We discovered that a novel SNP rs117565607_A at TRIM26 displayed the strongest association (OR = 1.909, Pcombined = 2.750 × 10-19 ). We also observed that TRIM26 was significantly downregulated in NPC tissue samples with genotype AA/AT than TT. Immunohistochemistry (IHC) test also found the TRIM26 protein expression in NPC tissue samples with the genotype AA/AT was lower than TT. According to computational prediction, rs117565607 locus was a binding site for the transcription factor Yin Yang 1 (YY1). We observed that the luciferase activity of YY1 which is binding to the A allele of rs117565607 was suppressed. ChIP data showed that YY1 was binding with T not A allele. Significance analysis of microarray suggested that TRIM26 downregulation was related to low immune response in NPC. We have identified a novel gene TRIM26 and a novel SNP rs117565607_A associated with NPC risk by regulating transcriptional process and established a new functional link between TRIM26 downregulation and low immune response in NPC.

A novel three-dimensional tumorsphere culture system for the efficient and low-cost enrichment of cancer stem cells with natural polymers.

Cancer stem cells (CSCs) are considered to serve a key role in tumor progression, recurrence and metastasis. Tumorsphere culture is the most important method for enriching CSCs and is widely used in basic research and drug screening. However, the traditional suspension cell culture system has several disadvantages, including low efficiency, high cost and difficult procedure, making it difficult to produce tumorspheres on a large scale. In the present study, two biomaterials, methylcellulose (MC) and gellan gum (GG), were used to construct a novel culture system based on the traditional system. Subsequently, the characteristics of the novel three-dimensional (3D) culture system were evaluated, the design scheme was optimized, and the morphological and biological features of the tumorspheres cultured in this 3D system were compared with the traditional system. The results revealed that the tumorspheres cultured in the novel 3D system presented a higher seeding density and improved morphology, while maintaining stem-like properties. This evidence suggests that a simple, efficient and low-cost culture system that produces tumorspheres on a large scale was successfully constructed, which can be widely used in various aspects of stem cell research.

Klf4 reduces stemness phenotype, triggers mesenchymal-epithelial transition (MET)-like molecular changes, and prevents tumor progression in nasopharygeal carcinoma.

The reprogramming factor Krüppel-like factor 4 (Klf4), one of the Yamanaka's reprogramming factors, plays an essential role in reprogramming somatic cells into induced pluripotent stem cells (iPSCs). Klf4 is dysregulated and displays divergent functions in multiple malignancies, but the biological roles of Klf4 in nasopharyngeal carcinoma (NPC) remain unknown. The present study revealed that Klf4 downregulation in a cohort of human NPC biopsies is significantly associated with invasive and metastatic phenotypes of NPC. Our results showed exogenous expression of Klf4 significantly inhibited cell proliferation, decreased stemness, triggered mesenchymal-epithelial transition (MET)-like molecular changes, and suppressed migration and invasion of NPC cells, whereas depletion of endogeneous Klf4 by RNAi reversed the aforementioned biological behaviors and characheristics. Klf4 silencing significantly enhanced the metastatic ability of NPC cells in vivo. In addition, CHIP assay confirmed that E-cadherin is a transcriptional target of Klf4 in NPC cells. Additional studies demonstrated that Klf4-induced MET-like cellular marker alterations, and reduced motility and invasion of NPC cells were mediated by E-cadherin. This study revealed the clinical correlation between Klf4 expression and epithelial-mesenchymal transition (EMT) biomarkers (including its target gene E-cadherin) in a cohort of NPC biopsies. Taken together, our findings suggest, for what we believe is the first time, that Klf4 functions as a tumor suppressor in NPC to decrease stemness phenotype, inhibit EMT and prevent tumor progression, suggesting that restoring Klf4 function may provide therapeutic benefits in NPC.

5T4-specific chimeric antigen receptor modification promotes the immune efficacy of cytokine-induced killer cells against nasopharyngeal carcinoma stem cell-like cells.

Relapse and metastasis of nasopharyngeal carcinoma (NPC) are presumably attributed to cancer stem cells (CSCs). In recent years, chimeric antigen receptor (CAR)-modified immune effector cells have been shown to have impressive antitumour efficacy. In this study, we aimed to identify appropriate tumour-associated antigens predominantly expressed on NPC stem cells (NPCSCs) and determine their suitability for CAR-engineered cytokine-induced killer (CIK) cell therapy against NPC. By investigating the expression patterns of potential target antigens (ROR1, 5T4 and CAIX) in NPC, we found that the oncofetal antigen 5T4 was predominately expressed in NPC cell lines and tissues but absent in non-cancerous nasopharyngeal tissues. Moreover, significantly enhanced expression of 5T4 in NPC spheroids revealed its relationship with putative NPCSCs. Hence, we designed a CAR construct (5T4-28Z) specific for 5T4 and generated CAR-transduced CIK cells. Our results showed that the artificial CAR was efficiently expressed on the surface of CIK cells and that no native phenotypes were altered by the gene transduction. Functional assays revealed that 5T4-28Z-CIK cells possessed both CAR-mediated and CAR-independent anti-NPC activity and were capable of efficiently attacking NPC cells, especially NPCSC-like cells in vitro, suggesting that they might serve as an attractive tool for developing efficient therapies against NPC.