GSTM3 enhances radiosensitivity of nasopharyngeal carcinoma by promoting radiation-induced ferroptosis through USP14/FASN axis and GPX4.

BACKGROUND: Radiotherapy is a critical treatment modality for nasopharyngeal carcinoma (NPC). However, the mechanisms underlying radiation resistance and tumour recurrence in NPC remain incompletely understood. METHODS: Oxidised lipids were assessed through targeted metabolomics. Ferroptosis levels were evaluated using cell viability, clonogenic survival, lipid peroxidation, and transmission electron microscopy. We investigated the biological functions of glutathione S-transferase mu 3 (GSTM3) in cell lines and xenograft tumours. Co-immunoprecipitation, mass spectrometry, and immunofluorescence were conducted to explore the molecular mechanisms involving GSTM3. Immunohistochemistry was performed to investigate the clinical characteristics of GSTM3. RESULTS: Ionising radiation (IR) promoted lipid peroxidation and induced ferroptosis in NPC cells. GSTM3 was upregulated following IR exposure and correlated with IR-induced ferroptosis, enhancing NPC radiosensitivity in vitro and in vivo. Mechanistically, GSTM3 stabilised ubiquitin-specific peptidase 14 (USP14), thereby inhibiting the ubiquitination and subsequent degradation of fatty acid synthase (FASN). Additionally, GSTM3 interacted with glutathione peroxidase 4 (GPX4) and suppressed GPX4 expression. Combining IR treatment with ferroptosis inducers synergistically improved NPC radiosensitivity and suppressed tumour growth. Notably, a decrease in GSTM3 abundance predicted tumour relapse and poor prognosis. CONCLUSIONS: Our findings elucidate the pivotal role of GSTM3 in IR-induced ferroptosis, offering strategies for the treatment of radiation-resistant or recurrent NPC.

Senescence-Related lncRNA Signature Predicts Prognosis, Response to Immunotherapy and Chemotherapy in Skin Cutaneous Melanoma.

Skin cutaneous melanoma (SKCM) is a highly malignant and aggressive cancer. Previous studies have shown that cellular senescence is a promising therapeutic strategy to limit melanoma cell progression. However, models to predict the prognosis of melanoma based on senescence-related lncRNAs and the efficacy of immune checkpoint therapy remain undefined. In this study, we developed a predictive signature consisting of four senescence-related lncRNAs (AC009495.2, U62317.1, AATBC, MIR205HG), and we then classified patients into high- and low-risk groups. GSEA (Gene set enrichment analysis) showed different activation of immune-related pathways in two groups. In addition, there were significant differences between the scores of tumor immune microenvironment, tumor burden mutation, immune checkpoint expression, and chemotherapeutic drug sensitivity between the two groups of patients. It provides new insights to guide more personalized treatment for patients with SKCM.

Case report: Abolishing primary resistance to PD-1 blockade by short-term treatment of lenvatinib in a patient with advanced metastatic renal cell carcinoma.

Anti-PD-1 immunotherapy has been extensively used in treatment of patients with advanced metastatic renal cell carcinoma (mRCC). Several prospective clinical trials showed that the combined treatment of anti-PD-1 antibody plus lenvatinib, a potent receptor tyrosine kinase inhibitor (TKI), exhibited high response rate compared with single-agent sunitinib. However, whether the patients with primary resistance to PD-1 blockade could benefit from the addition of lenvatinib is still unclear. Herein, we reported a patient with mRCC who was primary resistant to pembrolizumab and achieved a durable complete response after a short-term treatment with lenvatinib. This case report indicates that the patients with primary resistance to anti-PD-1 therapy could benefit from the short-term lenvatinib in combination with anti-PD-1 therapy, and provides a useful paradigm worthy of establishing a clinical trial for mRCC patients with primary resistance to anti-PD-1 therapy.

CYLD deficiency enhances metabolic reprogramming and tumor progression in nasopharyngeal carcinoma via PFKFB3.

Aberrant cancer metabolism contributes to cell proliferation and tumor progression. However, the contribution of enhanced glycolysis, observed during cancer metabolism, to the pathogenesis and progression of nasopharyngeal carcinoma (NPC) remains unclear. CYLD, an NF-κB inhibitor, is frequently deficient in NPC. Here, we investigated the role of CYLD in the metabolic reprogramming of NPC and found that restoration of CYLD expression suppressed glycolysis in NPC cells. Mechanistic dissection showed that CYLD stabilized p53 and facilitated its nuclear translocation, thereby enhancing p53 activity by removing K63-linked and K48-linked ubiquitin chains of p53, which can bind to the PFKFB3 promoter and inhibit its transcription. Additionally, CYLD interacted with FZR1 to promote APC/C-FZR1 E3 ligase activity, which further ubiquitinated and degraded PFKFB3 via the 26S proteasomal system. Furthermore, clinical tissue array analysis indicated that low expression of CYLD was correlated with high expression of PFKFB3 and poor prognosis among patients with NPC. In conclusion, CYLD suppressed PFKFB3 expression via two factors, namely, p53 and FZR1, to inhibit glycolysis and delay tumor growth and progression in NPC. CYLD is a biomarker indicating poor prognosis of patients with NPC.

Protein tyrosine phosphatase receptor type D gene promotes radiosensitivity via STAT3 dephosphorylation in nasopharyngeal carcinoma.

Radiotherapy is essential to the treatment of nasopharyngeal carcinoma (NPC) and acquired or innate resistance to this therapeutic modality is a major clinical problem. However, the underlying molecular mechanisms in the radiation resistance in NPC are not fully understood. Here, we reanalyzed the microarray data from public databases and identified the protein tyrosine phosphatase receptor type D (PTPRD) as a candidate gene. We found that PTPRD was downregulated in clinical NPC tissues and NPC cell lines with its promoter hypermethylated. Functional assays revealed that PTPRD overexpression sensitized NPC to radiation in vitro and in vivo. Importantly, miR-454-3p directly targets PTPRD to inhibit its expression and biological effect. Interestingly, mechanistic analyses indicate that PTPRD directly dephosphorylates STAT3 to enhance Autophagy-Related 5 (ATG5) transcription, resulting in triggering radiation-induced autophagy. The immunohistochemical staining of 107 NPC revealed that low PTPRD and high p-STAT3 levels predicted poor clinical outcome. Overall, we showed that PTPRD promotes radiosensitivity by triggering radiation-induced autophagy via the dephosphorylation of STAT3, thus providing a potentially useful predictive biomarker for NPC radiosensitivity and drug target for NPC radiosensitization.

Epstein-Barr virus (EBV) encoded microRNA BART8-3p drives radioresistance-associated metastasis in nasopharyngeal carcinoma.

Radiotherapy plays an important role in the treatment of nasopharyngeal carcinoma (NPC), however, 20% of patients with NPC exhibit unusual radioresistance. Patients with radioresistance are at risk of recurrence, so it is imperative to explore the mechanism of resistance to radiotherapy. In the past, studies on the mechanism of radioresistance have been restricted to DNA damage and related cell cycle remodeling or apoptosis. So far, no studies have explored the relationship between radioresistance and metastasis. Through the analysis of clinical samples, we observed that the metastasis rate of recurrent NPC was much higher than that of primary patients. In vitro and in vivo experiments showed that NPC cells with acquired radioresistance exhibited a stronger ability for invasion and metastasis. Mechanistically, we found that the Epstein-Barr virus (EBV)-encoded miRNA BART8-3p was increased in patients with NPC, and its expression was positively correlated with adverse prognostic factors, such as radioresistance. Besides this, miR-BART8-3p promoted the epithelial-mesenchymal transition, invasion, and metastasis of radioresistant NPC cells by targeting and inhibiting their PAG1 host gene. These findings suggested a novel role for EBV-miR-BART8-3p in promoting NPC radioresistance-associated metastasis and highlighted its potential value as a prognostic indicator or therapeutic target.

Multi-modal magnetic resonance imaging-based grading analysis for gliomas by integrating radiomics and deep features.

BACKGROUND: To investigate the feasibility of integrating global radiomics and local deep features based on multi-modal magnetic resonance imaging (MRI) for developing a noninvasive glioma grading model. METHODS: In this study, 567 patients [211 patients with glioblastomas (GBMs) and 356 patients with low-grade gliomas (LGGs)] between May 2006 and September 2018, were enrolled and divided into training (n=186), validation (n=47), and testing cohorts (n=334), respectively. All patients underwent postcontrast enhanced T1-weighted and T2 fluid-attenuated inversion recovery MRI scanning. Radiomics and deep features (trained by 8,510 3D patches) were extracted to quantify the global and local information of gliomas, respectively. A kernel fusion-based support vector machine (SVM) classifier was used to integrate these multi-modal features for grading gliomas. The performance of the grading model was assessed using the area under receiver operating curve (AUC), sensitivity, specificity, Delong test, and t-test. RESULTS: The AUC, sensitivity, and specificity of the model based on combination of radiomics and deep features were 0.94 [95% confidence interval (CI): 0.85, 0.99], 86% (95% CI: 64%, 97%), and 92% (95% CI: 75%, 99%), respectively, for the validation cohort; and 0.88 (95% CI: 0.84, 0.91), 88% (95% CI: 80%, 93%), and 81% (95% CI: 76%, 86%), respectively, for the independent testing cohort from a local hospital. The developed model outperformed the models based only on either radiomics or deep features (Delong test, both of P<0.001), and was also comparable to the clinical radiologists. CONCLUSIONS: This study demonstrated the feasibility of integrating multi-modal MRI radiomics and deep features to develop a promising noninvasive grading model for gliomas.

CYLD Promotes Apoptosis of Nasopharyngeal Carcinoma Cells by Regulating NDRG1.

PURPOSE: Nasopharyngeal carcinoma (NPC) is among the most common malignancies derived from the epithelium of the nasopharynx. To date, the regulatory networks involved in NPC have not been fully identified. Previous studies revealed multiple loss-of-function mutations in NPC and specifically in cylindromatosis lysine 63 deubiquitinase (CYLD); however, the exact role of CYLD in NPC progression and its potential mechanism remains unclear. METHODS: We performed immunohistochemical (IHC) staining and real-time quantitative polymerase chain reaction (qPCR) to measure CYLD expression in NPC tissues, and Western blot was conducted to determine CYLD levels in NPC cell lines. Cell proliferation was detected by CCK8 assay and colony formation analysis, and apoptosis was determined by Annexin V/propidium iodide staining. Potential targets of CYLD were verified by co-immunoprecipitation and mass spectrometry. Xenograft assay was conducted to confirm the role of CYLD in vivo. RESULTS: We found that CYLD levels were significantly decreased in both NPC tissues and cell lines, and that CYLD overexpression inhibited NPC cell proliferation and promoted apoptosis. Additionally, we revealed that CYLD bound and upregulated N-Myc downstream regulated 1 (NDRG1), and that silencing NDRG1 abolished the tumor-suppressor effect of CYLD on NPC cells. Furthermore, CYLD suppressed tumor growth in xenograft mice models. CONCLUSION: These results suggest CYLD as a tumor suppressor, potential biomarker for diagnosing NPC, and therapeutic target.

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.

Influence and mechanism of miR-99a suppressing development of colorectal cancer (CRC) with diabetes mellitus (DM).

OBJECTIVE: This study aimed to identify the changes of miRNAs in colorectal cancer (CRC) complicated with diabetes mellitus (DM) (CRC + DM) tissues and their potential effects. METHODS: The changes of miRNAs in CRC + DM tissues were determined by miRNA microarray. The expression levels of miR-99a in 40 clinical specimens and 6 CRC cell lines were determined by qRT-PCR. The capacity for miR-99a to induce cell proliferation and invasion was examined with miR-99a-overexpressing HCT-116 cells. The relative mTOR mRNA and protein levels were determined by qRT-PCR and Western blotting, respectively, in HCT-116 cells transfected with miR-99a. The dual luciferase assay was performed to confirm the direct regulation of miR-99a on mTOR 3'-UTR. The HCT-116 cells were treated with 100 mg/L advanced glycation end products (AGEs); then, the mTOR expression levels were determined by qRT-PCR, Western blotting, and immunohistochemistry. RESULTS: Seventeen miRNAs were found to be differentially expressed among normal tissue, CRC tissue, and CRC with DM tissue, including 15 upregulated and 2 downregulated with fold changs of more than 2 times. qRT-PCR confirmed that miR-99a was downregulated in CRC and CRC + DM tissues. In addition, miR-99a overexpression remarkably impaired CRC cell proliferation and metastasis, and negatively regulated mTOR signaling through direct binding to the 3'-UTR of mTOR. AGEs could suppress miR-99a and stimulate mTOR signaling in CRC cells. Increased mTOR was also identified in CRC with DM tissues. CONCLUSION: Our findings indicate that miR-99a is a potential marker and therapeutic target of CRC complicated with DM, and that AGEs impair miR-99a-overactivated mTOR signaling in CRC with DM patients, which promotes CRC development.

EBV-miR-BART7-3p Imposes Stemness in Nasopharyngeal Carcinoma Cells by Suppressing SMAD7.

Cancer stem-like cells, possessing "stemness" properties, play crucial roles in progression, metastasis, and drug resistance in various cancers. Viral microRNAs (such as EBV-miR-BART7-3p), as exogenous regulators, have been discovered to regulate malignant progression of nasopharyngeal carcinoma (NPC), suggesting a possible role of viral microRNAs in imposing stemness. In this study, we found that EBV-miR-BART7-3p induce stemness of NPC cells. We firstly reported that EBV-miR-BART7-3p increased the percentage of side population cells, the development of tumor spheres, and the expression level of stemness markers in vitro. This viral microRNA also enhanced stem-like or cancer-initiating properties of NPC cells in vivo. Besides, we identified SMAD7 as a novel target gene of EBV-miR-BART7-3p in addition to PTEN gene we previously reported; this viral microRNA suppressed SMAD7, led to activation of TGF-ß signaling, and eventually enhanced the stemness of NPC cells. Silencing of SMAD7 resembled the effects generated by EBV-miR-BART7-3p in NPC cells. After reconstitution of SMAD7, EBV-miR-BART7-3p-expressing cells underwent a phenotypic reversion. EBV-positive NPC cells were used to enable experimental validation. Finally, we further discovered that EBV-miR-BART7-3p increased chemo-resistance of NPC in vitro and in vivo, supporting that EBV-miR-BART7-3 resulted in increased stemness of NPC cells and lead to drug resistance and cancer recurrence. Overall, this study uncovered a novel mechanism underlying viral microRNA-associated stemness of NPC cells. This viral microRNA and its associated cellular genes may be potential therapeutic targets for restraining chemo-resistance and recurrence of NPC.

EBV encoded miRNA BART8-3p promotes radioresistance in nasopharyngeal carcinoma by regulating ATM/ATR signaling pathway.

Resistance to radiotherapy is one of the main causes of treatment failure in patients with nasopharyngeal carcinoma (NPC). Epstein-Barr virus (EBV) infection is an important factor in the pathogenesis of NPC, and EBV-encoded microRNAs (miRNAs) promote NPC progression. However, the role of EBV-encoded miRNAs in the radiosensitivity of NPC remains unclear. Here, we investigated the effects of EBV-miR-BART8-3p on radiotherapy resistance in NPC cells in vitro and in vivo, and explored the underlying molecular mechanisms. Inhibitors of ataxia telangiectasia mutated (ATM)/ataxia telangiectasia mutated and Rad3-related (ATR) (KU60019 and AZD6738, respectively) were used to examine radiotherapy resistance. We proved that EBV-miR-BART8-3p promoted NPC cell proliferation in response to irradiation in vitro and associated with the induction of cell cycle arrest at the G2/M phase, which was a positive factor for the DNA repair after radiation treatment. Besides, EBV-miR-BART8-3p could increase the size of xenograft tumors significantly in nude mice. Treatment with KU60019 or AZD6738 increased the radiosensitivity of NPC by suppressing the expression of p-ATM and p-ATR. The present results indicate that EBV-miR-BART8-3p promotes radioresistance in NPC by modulating the activity of ATM/ATR signaling pathway.

S100A4 suppresses cancer stem cell proliferation via interaction with the IKK/NF-κB signaling pathway.

BACKGROUND: Bladder cancer often recurs due to incomplete elimination of the cancer stem cells (CSCs). Therefore, new strategies targeting bladder CSCs are needed and the aim of this study was to investigate the effect of S100A4 on the proliferation capacity of MB49 bladder cancer stem cells (MCSCs). METHODS: MCSCs were established and validated. The expression level of S100A4 in MCSCs and MB49 cells was evaluated using Western blotting and quantitative polymerase chain reaction (QPCR). S100A4 was overexpressed or knocked-down by transfection of pCMV6-XL5-S100A4 plasmid or RNA interference (RNAi) respectively. Proliferation capacity of MCSC was evaluated by cell proliferation assay and in vivo tumorigenicity study. Transcriptional activity of nuclear factor kappa B (NF-κB) was analyzed using luciferase reporter assay, and the level of interleukin (IL)-2 as well as tumor necrosis factor (TNF) was quantified by QPCR. Protein-protein interaction of S100A4 and inhibitor of nuclear factor kappa B NF-κB kinase (IKK) was analyzed by immunoprecipitation. RESULTS: S100A4 was significantly up-regulated in MCSCs, which positively associated with the proliferation capacity, as well as the level of NF-κB, IKK, IL-2 and TNF in MCSCs. Knock-down of S100A4 could reverse such effects. Using immunoprecipitation assay, an interaction between S100A4 and IKK could be observed. CONCLUSIONS: S100A4 is upregulated in MCSCs and possibly enhance the proliferation ability of MCSCs by way of activating the IKK/NF-κB signaling pathway, and S100A4 maybe a hopeful therapeutic target for MCSCs.

Influence and mechanism of 5-aminolevulinic acid-photodynamic therapy on the metastasis of esophageal carcinoma.

BACKGROUD: Photodynamic therapy (PDT) for the treatment of esophageal cancer was more and more popularly used since it was approved for the treatment of advanced esophageal cancer in 1996. It has been reported to influence the tumor growth and metastasis via a variety of signaling pathways, but its mechanism remains to be further studied. This research studied the effects of ALA-PDT on esophageal carcinoma in vitro and in vivo, discovering its molecular regulating mechanism and the way to enhence the PDT effect. METHODS: Eca-109 cells were incubated with a medium containing EGFR tyrphostin AG1478 or PI3K inhibitor LY294002, then with ALA, and the cells were irradiated with the laser 6h later. The cell viability was measured with MTT assay, and the migration ability was detected by transwell experiments 24h post-ALA-PDT. The gene and protein expression on EGFR/PI3K/AKT signaling pathway was analyzed by realtime PCR and Western blotting respectively. Then, RFP-Eca-109 burdened nude mice model was constructed, and were treated with ALA-PDT when the tumor volume reached 150-350mm3. The gene and protein expression were analyzed 24h and 50days post-ALA-PDT. RESULTS: Our study showed that ALA-PDT respectively combined with AG1478, LY294002 could synergistically reduce the growth and migration ability of the Eca-109 cells in vitro and significantly down-regulate the protein expression of EGFR/PI3K and PI3K/AKT, meanwhile, significantly down-regulate the gene expression of EGFR when combining with AG1478. Forthermore, ALA-PDT could significantly decrease the tumor growth and metastasis and down-regulate the gene expression of EGFR and the protein expression of EGFR and PI3K in the tumor of mice. CONCLUSION: This study revealed a molecular mechanism of ALA-PDT and developed a new modality application of therapy, by combining ALA-PDT with small molecular inhibitors, for better effect in the clinical practice of esophageal carcinoma.

Prognostic impact of pretherapeutic gamma-glutamyltransferase on patients with nasopharyngeal carcinoma.

BACKGROUND: Gamma-glutamyltransferase (GGT) is a membrane-bound enzyme involved in the metabolism of glutathione. Studies suggested that GGT played an important role in the tumor development, progression, invasion and drug resistance and prognosis. The association between GGT and prognosis of patients with nasopharyngeal carcinoma (NPC) was unknown. This study was conducted to investigate the association of pretherapeutic serum level of GGT with clinical-pathological parameters and survival in patients with NPC. METHODS: Two hundred and twenty-two patients with NPC were recruited in this study and were stratified into two GGT risk groups (≤ 34.5 U/L, > 34.5 U/L). The association of pretherapeutic serum GGT levels with clinical-pathological parameters was examined. Univariate and multivariate survival analyses were performed. FINDINGS: The pretherapeutic serum level of GGT was not associated with gender, age, pathology, T stage, N stage, TNM stage, chemotherapy or radiotherapy in patients with NPC. Patients in the high-risk GGT group had a poorer survival than the low-risk GGT group (3-year overall survival, 74.2% vs. 50.2%, P = 0.001; 3-year progression-free survival, 76.4% vs. 47.1%, P < 0.001; 3-year loco-regional relapse-free survival, 76.4% vs. 51.3%, P < 0.001; 3-year distant metastasis-free survival, 89.5% vs. 66.4%, P < 0.001). Multivariate analysis suggested that patients in the high-risk GGT group had 2.117 (95% confidence interval [CI], 1.225 ∼ 3.659, P = 0.007) times the risk of death, 2.836 (95% CI, 1.765 ∼ 4.557, P < 0.001) times the risk of progression, 2.551 (95% CI, 1.573 ∼ 4.138, P < 0.001) times the risk of relapse, and 3.331 (95% CI, 1.676 ∼ 6.622, P < 0.001) times the risk of metastasis compared with those in the low-risk GGT group. CONCLUSION: The pretherapeutic serum level of GGT might serve as a novel independent prognostic factor for overall-survival, progression-free survival, loco-regional relapse-free survival and distant metastasis-free survival in patients with NPC.

miR156a Mimic Represses the Epithelial-Mesenchymal Transition of Human Nasopharyngeal Cancer Cells by Targeting Junctional Adhesion Molecule A.

MicroRNAs (miRNAs) have been documented as having an important role in the development of cancer. Broccoli is very popular in large groups of the population and has anticancer properties. Junctional adhesion molecule A (JAMA) is preferentially concentrated at tight junctions and influences cell morphology and migration. Epithelial-mesenchymal transition (EMT) is a developmental program associated with cancer progression and metastasis. In this study we aimed to investigate the role of miRNAs from broccoli in human nasopharyngeal cancer (NPC). We demonstrated that a total of 84 conserved miRNAs and 184 putative novel miRNAs were found in broccoli by sequencing technology. Among these, miR156a was expressed the most. In addition, synthetic miR156a mimic inhibited the EMT of NPC cells in vitro. Furthermore, it was confirmed that JAMA was the target of miR156a mimic as validated by 3' UTR luciferase reporter assays and western blotting. Knockdown of JAMA was consistent with the effects of miR156a mimic on the EMT of NPC, and the up-regulation of JAMA could partially restore EMT repressed by miR156a mimic. In conclusion, these results indicate that the miR156a mimic inhibits the EMT of NPC cells by targeting the 3' UTR of JAMA. These miRNA profiles of broccoli provide a fundamental basis for further research. Moreover, the discovery of miR156a may have clinical implications for the treatment of patients with NPC.

The important role of the receptor for activated C kinase 1 (RACK1) in nasopharyngeal carcinoma progression.

BACKGROUND: The receptor for activated C kinase 1 (RACK1) is involved in various cancers, but its roles in nasopharyngeal carcinoma (NPC) have not yet been fully elucidated. METHODS: Initially, RACK1 expression was analyzed by immunohistochemistry in NPC and normal nasopharyngeal (NP) tissues. It was also detected by qPCR and Western blot in NPC cells. Confocal microscope and immunofluorescence were performed to detect the subcellular compartmentalization of RACK1. Subsequently, after up- or down-regulating RACK1 in NPC cells, cell proliferation and migration/invasion were tested using in vitro assays including MTT, EdU, colony formation, Transwell and Boyden assays. Furthermore, several key molecules were detected by Western blot to explore underlying mechanism. Finally, clinical samples were analyzed to confirm the relationship between RACK1 expression and clinical features. RESULTS: Receptor for activated C kinase 1 expression was much higher in NPC than NP tissues. And RACK1 was mainly located in the cytoplasm. Overexpression of RACK1 promoted NPC cell proliferation and metastasis/invasion, whereas depletion of this protein suppressed NPC cell proliferation and metastasis/invasion. Mechanistically, RACK1 deprivation obviously suppressed the activation of Akt and FAK, suggesting the PI3K/Akt/FAK pathway as one of functional mechanisms of RACK1 in NPC. Furthermore, clinical sample analysis indicated a positive correlation between in vivo expression of RACK1 with lymph node invasion and clinical stage of NPC. CONCLUSION: Our results demonstrate that RACK1 protein plays an important role in NPC development and progression. The upregulation of RACK1 can promote the proliferation and invasion of NPC by regulating the PI3K/Akt/FAK signal pathway. Thus, this study contributes to the discovery of a potential therapeutic target for NPC.

Higher methylation intensity induced by EBV LMP1 via NF-κB/DNMT3b signaling contributes to silencing of PTEN gene.

Phosphatase and tensin homolog (PTEN) is a major tumor suppressor and usually silenced via the deletion, insertion and mutation. We previously discovered its inactivation via aberrant CpG island methylation. Here, we provide further evidence that EBV latent membrane protein 1(LMP1) can induce a higher intensity of DNA methylation at PTEN CpG islands, inactivating PTEN at the cellular and molecular level. Initially, increased methylation intensity of PTEN CpG islands was observed in EBV-infected nasopharyngeal carcinoma (NPC) cells, accompanied by decreased PTEN expression. In NPC tissue samples showing the methylation at PTEN promoter, LMP1 was highly expressed in higher methylation intensity group relative to lower intensity group, and DNA methyltransferase 3b (DNMT3b) expression was positively correlated with LMP1 expression. Moreover, transfection of LMP1 gene into EBV-negative NPC cells demonstrated that LMP1 up-regulated DNMT3b expression, leading to a higher intensity of PTEN CpG island methylation. Mechanistically, computational prediction and luciferase reporter assay identified a functional NF-κB binding site on DNMT3b promoter and the mutated NF-κB binding site abolished LMP1-mediated DNMT3b activation. Chromatin immunoprecipitation displayed that NF-κB p65 subunit constitutively bound to DNMT3b promoter, supporting the activation of DNMT3b by EBV LMP1 via NF-κB signaling. Furthermore, the expression level of DNMT3b was observed to be increased in the nuclei of LMP1-expressing NPC cells, and a NF-κB inhibitor, PDTC, counteracted LMP1-mediated DNMT3b overexpression. Thus, this study first reports that LMP1-mediated NF-κB can up-regulate DNMT3b transcription, thereby leading to relatively higher methylation intensity at PTEN CpG islands, and ultimately silencing major tumor suppressor PTEN.

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

Epstein-Barr virus (EBV), aetiologically linked to nasopharyngeal carcinoma (NPC), is the first human virus found to encode many miRNAs. However, how these viral miRNAs precisely regulate the tumour metastasis in NPC remains obscure. Here we report that EBV-miR-BART1 is highly expressed in NPC and closely associated with pathological and advanced clinical stages of NPC. Alteration of EBV-miR-BART1 expression results in an increase in migration and invasion of NPC cells in vitro and causes tumour metastasis in vivo. Mechanistically, EBV-miR-BART1 directly targets the cellular tumour suppressor PTEN. Reduction of PTEN dosage by EBV-miR-BART1 activates PTEN-dependent pathways including PI3K-Akt, FAK-p130(Cas) and Shc-MAPK/ERK1/2 signalling, drives EMT, and consequently increases migration, invasion and metastasis of NPC cells. Reconstitution of PTEN rescues all phenotypes generated by EBV-miR-BART1, highlighting the role of PTEN in EBV-miR-BART-driven metastasis in NPC. Our findings provide new insights into the metastasis of NPC regulated by EBV and advocate for developing clinical intervention strategies against NPC.

Gold nano-particles (AuNPs) carrying anti-EBV-miR-BART7-3p inhibit growth of EBV-positive nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) infection is a major etiological factor for nasopharyngeal carcinoma (NPC). Several EBV-encoded BART miRNAs have been associated with viral latency, immune escape, cell survival, cell proliferation and apoptosis. Here, we report that EBV-miR-BART7-3p, an EBV-encoded BART miRNA highly expressed in NPC, was correlated with cell-cycle progression in vitro and increased tumor formation in vivo. This viral miRNA stimulated the PTEN/PI3K/Akt pathway and induced c-Myc and c-Jun. Knockdown of PTEN mimicked EBV-miR-BART7-3p-induced tumorigenic phenotype. Based on these results, we conducted a therapeutic experiment by using gold nano-particles (AuNPs) carrying anti-EBV-miR-BART7-3p. Silencing of EBV-miR-BART7-3p reduced tumor growth in animal model. We conclude that EBV-miR-BART7-3p favors carcinogenesis, representing a potential target for miRNA-based therapy.