Erratum: CHL1 suppresses tumor growth and metastasis in nasopharyngeal carcinoma by repressing PI3K/AKT signaling pathway via interaction with Integrin ß1 and Merlin: Erratum.

[This corrects the article DOI: 10.7150/ijbs.34785.].

M6PR- and EphB4-Rich Exosomes Secreted by Serglycin-Overexpressing Esophageal Cancer Cells Promote Cancer Progression.

Accumulating evidence shows that exosomes participate in cancer progression. However, the functions of cancer cell exosome-transmitted proteins are rarely studied. Previously, we reported that serglycin (SRGN) overexpression promotes invasion and metastasis of esophageal squamous cell carcinoma (ESCC) cells. Here, we investigated the paracrine effects of exosomes from SRGN-overexpressing ESCC cells (SRGN Exo) on ESCC cell invasion and tumor angiogenesis, and used mass spectrometry to identify exosomal proteins involved. Cation-dependent mannose-6-phosphate receptor (M6PR) and ephrin type-B receptor 4 (EphB4) were pronouncedly upregulated in SRGN Exo. Upregulated exosomal M6PR mediated the pro-angiogenic effects of SRGN Exo both in vitro and in vivo, while augmented exosomal EphB4 mediated the pro-invasive effect of SRGN Exo on ESCC cells in vitro. In addition, in vitro studies showed that manipulation of M6PR expression affected the viability and migration of ESCC cells. Both M6PR and EphB4 expression levels were positively correlated with that of SRGN in the serum of patients with ESCC. High level of serum M6PR was associated with poor overall survival rates. Taken together, this study presents the first proof that exosomal M6PR and EphB4 play essential roles in tumor angiogenesis and malignancy, and that serum M6PR is a novel prognostic marker for ESCC patients.

Latent membrane protein 1 and macrophage-derived TNFα synergistically activate and mobilize invadopodia to drive invasion of nasopharyngeal carcinoma.

Invadopodia are actin-rich membrane protrusions that digest the matrix barrier during cancer metastasis. Since the discovery of invadopodia, they have been visualized as localized and dot-like structures in different types of cancer cells on top of a 2D matrix. In this investigation of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), a highly invasive cancer frequently accompanied by neck lymph node and distal organ metastases, we revealed a new form of invadopodium with mobilizing features. Integration of live-cell imaging and molecular assays revealed the interaction of macrophage-released TNFα and EBV-encoded latent membrane protein 1 (LMP1) in co-activating the EGFR/Src/ERK/cortactin and Cdc42/N-WASP signaling axes for mobilizing the invadopodia with lateral movements. This phenomenon endows the invadopodia with massive degradative power, visualized as a shift of focal dot-like digestion patterns on a 2D gelatin to a dendrite-like digestion pattern. Notably, single stimulation of either LMP1 or TNFα could only enhance the number of ordinary dot-like invadopodia, suggesting that the EBV infection sensitizes the NPC cells to form mobilizing invadopodia when encountering a TNFα-rich tumor microenvironment. This study unveils the interplay of EBV and stromal components in driving the invasive potential of NPC via unleashing the propulsion of invadopodia in overcoming matrix hurdles. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The microdissected gene expression landscape of nasopharyngeal cancer reveals vulnerabilities in FGF and noncanonical NF-κB signaling.

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)-positive epithelial malignancy with an extensive inflammatory infiltrate. Traditional RNA-sequencing techniques uncovered only microenvironment signatures, while the gene expression of the tumor epithelial compartment has remained a mystery. Here, we use Smart-3SEQ to prepare transcriptome-wide gene expression profiles from microdissected NPC tumors, dysplasia, and normal controls. We describe changes in biological pathways across the normal to tumor spectrum and show that fibroblast growth factor (FGF) ligands are overexpressed in NPC tumors, while negative regulators of FGF signaling, including SPRY1, SPRY2, and LGALS3, are down-regulated early in carcinogenesis. Within the NF-κB signaling pathway, the critical noncanonical transcription factors, RELB and NFKB2, are enriched in the majority of NPC tumors. We confirm the responsiveness of EBV-positive NPC cell lines to targeted inhibition of these pathways, reflecting the heterogeneity in NPC patient tumors. Our data comprehensively describe the gene expression landscape of NPC and unravel the mysteries of receptor tyrosine kinase and NF-κB pathways in NPC.

Exosomes derived from γδ-T cells synergize with radiotherapy and preserve antitumor activities against nasopharyngeal carcinoma in immunosuppressive microenvironment.

BACKGROUND: Radiotherapy is the first-line treatment for patients nasopharyngeal carcinoma (NPC), but its therapeutic efficacy is poor in some patients due to radioresistance. Adoptive T cell-based immunotherapy has also shown promise to control NPC; however, its antitumor efficacy may be attenuated by an immunosuppressive tumor microenvironment. Exosomes derived from γδ-T cells (γδ-T-Exos) have potent antitumor potentials. However, it remains unknown whether γδ-T-Exos have synergistic effect with radiotherapy and preserve their antitumor activities against NPC in an immunosuppressive tumor microenvironment. METHODS: γδ-T-Exos were stained with fluorescent membrane dye, and their interactions with NPC were determined both in vitro and in vivo. NPC cell deaths were detected after treatment with γδ-T-Exos and/or irradiation. Moreover, effects of γδ-T-Exos on radioresistant cancer stem-like cells (CSCs) were determined. The therapeutic efficacy of combination therapy using γδ-T-Exos and irradiation on NPC tumor progression was also monitored in vivo. Finally, the tumor-killing and T cell-promoting activities of γδ-T-Exos were determined under the culture in immunosuppressive NPC supernatant. RESULTS: γδ-T-Exos effectively interacted with NPC tumor cells in vitro and in vivo. γδ-T-Exos not only killed NPC cells in vitro, which was mainly mediated by Fas/Fas ligand (FasL) and death receptor 5 (DR5)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) pathways, but also controlled NPC tumor growth and prolonged tumor-bearing mice survival in vivo. Furthermore, γδ-T-Exos selectively targeted the radioresistant CD44+/high CSCs and induced profound cell apoptosis. The combination of γδ-T-Exos with radiotherapy overcame the radioresistance of CD44+/high NPC cells and significantly improved its therapeutic efficacy against NPC in vitro and in vivo. In addition, γδ-T-Exos promoted T-cell migration into NPC tumors by upregulating CCR5 on T cells that were chemoattracted by CCR5 ligands in the NPC tumor microenvironment. Although NPC tumor cells secreted abundant tumor growth factor beta to suppress T-cell responses, γδ-T-Exos preserved their direct antitumor activities and overcame the immunosuppressive NPC microenvironment to amplify T-cell antitumor immunity. CONCLUSIONS: γδ-T-Exos synergized with radiotherapy to control NPC by overcoming the radioresistance of NPC CSCs. Moreover, γδ-T-Exos preserved their tumor-killing and T cell-promoting activities in the immunosuppressive NPC microenvironment. This study provides a proof of concept for a novel and potent strategy by combining γδ-T-Exos with radiotherapy in the control of NPC.

Longitudinal evaluation of five nasopharyngeal carcinoma animal models on the microPET/MR platform.

PURPOSE: We longitudinally evaluated the tumour growth and metabolic activity of three nasopharyngeal carcinoma (NPC) cell line models (C666-1, C17 and NPC43) and two xenograft models (Xeno76 and Xeno23) using a micropositron emission tomography and magnetic resonance (microPET/MR). With a better understanding of the interplay between tumour growth and metabolic characteristics of these NPC models, we aim to provide insights for the selection of appropriate NPC cell line/xenograft models to assist novel drug discovery and evaluation. METHODS: Mice were imaged by 18F-deoxyglucose ([18F]FDG) microPET/MR twice a week for consecutive 3-7 weeks. [18F]FDG uptake was quantified by standardized uptake value (SUV) and presented as SUVmean tumour-to-liver ratio (SUVRmean). Longitudinal tumour growth patterns and metabolic patterns were recorded. SUVRmean and histological characteristics were compared across the five NPC models. Cisplatin was administrated to one selected optimal tumour model, C17, to evaluate our imaging platform. RESULTS: We found variable tumour growth and metabolic patterns across different NPC tumour types. C17 has an optimal growth rate and higher tumour metabolic activity compared with C666-1. C666-1 has a fast growth rate but is low in SUVRmean at endpoint due to necrosis as confirmed by H&E. NPC43 and Xeno76 have relatively slow growth rates and are low in SUVRmean, due to severe necrosis. Xeno23 has the slowest growth rate, and a relative high SUVRmean. Cisplatin showed the expected therapeutic effect in the C17 model in marked reduction of tumour size and metabolism. CONCLUSION: Our study establishes an imaging platform that characterizes the growth and metabolic patterns of different NPC models, and the platform is well able to demonstrate drug treatment outcome supporting its use in novel drug discovery and evaluation for NPC.

ΔNp63α promotes Epstein-Barr virus latency in undifferentiated epithelial cells.

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and contributes to both B-cell and epithelial-cell malignancies. EBV-infected epithelial cell tumors, including nasopharyngeal carcinoma (NPC), are largely composed of latently infected cells, but the mechanism(s) maintaining viral latency are poorly understood. Expression of the EBV BZLF1 (Z) and BRLF1 (R) encoded immediate-early (IE) proteins induces lytic infection, and these IE proteins activate each other's promoters. ΔNp63α (a p53 family member) is required for proliferation and survival of basal epithelial cells and is over-expressed in NPC tumors. Here we show that ΔNp63α promotes EBV latency by inhibiting activation of the BZLF1 IE promoter (Zp). Furthermore, we find that another p63 gene splice variant, TAp63α, which is expressed in some Burkitt and diffuse large B cell lymphomas, also represses EBV lytic reactivation. We demonstrate that ΔNp63α inhibits the Z promoter indirectly by preventing the ability of other transcription factors, including the viral IE R protein and the cellular KLF4 protein, to activate Zp. Mechanistically, we show that ΔNp63α promotes viral latency in undifferentiated epithelial cells both by enhancing expression of a known Zp repressor protein, c-myc, and by decreasing cellular p38 kinase activity. Furthermore, we find that the ability of cis-platinum chemotherapy to degrade ΔNp63α contributes to the lytic-inducing effect of this agent in EBV-infected epithelial cells. Together these findings demonstrate that the loss of ΔNp63α expression, in conjunction with enhanced expression of differentiation-dependent transcription factors such as BLIMP1 and KLF4, induces lytic EBV reactivation during normal epithelial cell differentiation. Conversely, expression of ΔNp63α in undifferentiated nasopharyngeal carcinoma cells and TAp63α in Burkitt lymphoma promotes EBV latency in these malignancies.

A three-dimensional spheroid-specific role for Wnt-ß-catenin and Eph-ephrin signaling in nasopharyngeal carcinoma cells.

One of the greatest unmet needs hindering the successful treatment of nasopharyngeal carcinomas (NPCs) is for representative physiological and cost-effective models. Although Epstein-Barr virus (EBV) infection is consistently present in NPCs, most studies have focused on EBV-negative NPCs. For the first time, we established and analyzed three-dimensional (3D) spheroid models of EBV-positive and EBV-negative NPC cells and compared these to classical two-dimensional (2D) cultures in various aspects of tumor phenotype and drug responses. Compared to 2D monolayers, the 3D spheroids showed significant increases in migration capacity, stemness characteristics, hypoxia and drug resistance. Co-culture with endothelial cells, which mimics essential interactions in the tumor microenvironment, effectively enhanced spheroid dissemination. Furthermore, RNA sequencing revealed significant changes at the transcriptional level in 3D spheroids compared to expression in 2D monolayers. In particular, we identified known (VEGF, AKT and mTOR) and novel (Wnt-ß-catenin and Eph-ephrin) cell signaling pathways that are activated in NPC spheroids. Targeting these pathways in 3D spheroids using FDA-approved drugs was effective in monoculture and co-culture. These findings provide the first demonstration of the establishment of EBV-positive and EBV-negative NPC 3D spheroids with features that resemble advanced and metastatic NPCs. Furthermore, we show that NPC spheroids have potential use in identifying new drug targets.

Whole-genome profiling of nasopharyngeal carcinoma reveals viral-host co-operation in inflammatory NF-κB activation and immune escape.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer.

p85ß alters response to EGFR inhibitor in ovarian cancer through p38 MAPK-mediated regulation of DNA repair.

EGFR signaling promotes ovarian cancer tumorigenesis, and high EGFR expression correlates with poor prognosis. However, EGFR inhibitors alone have demonstrated limited clinical benefit for ovarian cancer patients, owing partly to tumor resistance and the lack of predictive biomarkers. Cotargeting EGFR and the PI3K pathway has been previously shown to yield synergistic antitumor effects in ovarian cancer. Therefore, we reasoned that PI3K may affect cellular response to EGFR inhibition. In this study, we revealed PI3K isoform-specific effects on the sensitivity of ovarian cancer cells to the EGFR inhibitor erlotinib. Gene silencing of PIK3CA (p110α) and PIK3CB (p110ß) rendered cells more susceptible to erlotinib. In contrast, low expression of PIK3R2 (p85ß) was associated with erlotinib resistance. Depletion of PIK3R2, but not PIK3CA or PIK3CB, led to increased DNA damage and reduced level of the nonhomologous end joining DNA repair protein BRD4. Intriguingly, these defects in DNA repair were reversed upon erlotinib treatment, which caused activation and nuclear import of p38 MAPK to promote DNA repair with increased protein levels of 53BP1 and BRD4 and foci formation of 53BP1. Remarkably, inhibition of p38 MAPK or BRD4 re-sensitized PIK3R2-depleted cells to erlotinib. Collectively, these data suggest that p38 MAPK activation and the subsequent DNA repair serve as a resistance mechanism to EGFR inhibitor. Combined inhibition of EGFR and p38 MAPK or DNA repair may maximize the therapeutic potential of EGFR inhibitor in ovarian cancer.

FBX4 mediates rapid cyclin D1 proteolysis upon DNA damage in immortalized esophageal epithelial cells.

It has been implied that deregulation of cyclin D1 turnover under stresses can facilitate genomic instability and trigger tumorigenesis. Much focus has been placed on identifying the E3 ligases responsible for mediating cyclin D1 degradation. However, the findings were quite controversial and cell type-dependent. Little is known about how cyclin D1 is regulated in precancerous cells upon DNA damage and which E3 ligases mediate the effects. Here we found cyclin D1 reduction is an early response to DNA damage in immortalized esophageal epithelial cells, with expression dropping to a low level within 1 h after γ-irradiation. Comparison of temporal expression of cyclin D1 upon DNA damage between immortalized NE083-hTERT and NE083-E6E7, the latter being p53/p21-defective, showed that DNA damage-induced rapid cyclin D1 reduction was p53-independent and occurred before p21 accumulation. Overexpression of cyclin D1 in NE083-E6E7 cells could attenuate G0/G1 cell cycle arrest at 1 h after irradiation. Furthermore, rapid reduction of cyclin D1 upon DNA damage was attributed to proteasomal degradation, as evidenced by data showing that proteasomal inhibition by MG132 blocked cyclin D1 reduction while cycloheximide facilitated it. Inhibition of ATM activation and knockdown of E3 ligase adaptor FBX4 reversed cyclin D1 turnover in immortalized NE083-hTERT cells. Further study showed that knockdown of FBX4 facilitated DNA breaks, as indicated by an increase in γ-H2AX foci in esophageal cancer cells. Taken together, the results substantiated a pivotal role of ATM and FBX4 in cyclin D1 proteolysis upon DNA damage in precancerous esophageal epithelial cells, implying that deregulation of the process may contribute to carcinogenesis of esophageal squamous cell carcinoma.

Dual-Targeting Peptide-Guided Approach for Precision Delivery and Cancer Monitoring by Using a Safe Upconversion Nanoplatform.

Using Epstein-Barr virus (EBV)-induced cancer cells and HeLa cells as a comparative study model, a novel and safe dual-EBV-oncoproteins-targeting pH-responsive peptide engineering, coating, and guiding approach to achieve precision targeting and treatment strategy against EBV-associated cancers is introduced. Individual functional peptide sequences that specifically bind to two overexpressed EBV-specific oncoproteins, EBNA1 (a latent cellular protein) and LMP1 (a transmembrane protein), are engineered in three different ways and incorporated with a pH-sensitive tumor microenvironment (TME)-cleavable linker onto the upconversion nanoparticles (UCNP) NaGdF4:Yb3+, Er3+@NaGdF4 (UCNP-P n , n = 5, 6, and 7). A synergistic combination of the transmembrane LMP1 targeting ability and the pH responsiveness of UCNP-P n is found to give specific cancer differentiation with higher cellular uptake and accumulation in EBV-infected cells, thus a lower dose is needed and the side effects and health risks from treatment would be greatly reduced. It also gives responsive UC signal enhancement upon targeted dual-protein binding and shows efficacious EBV cancer inhibition in vitro and in vivo. This is the first example of simultaneous imaging and inhibition of two EBV latent proteins, and serves as a blueprint for next-generation peptide-guided precision delivery nanosystem for the safe monitoring and treatment against one specific cancer.

Significance of serglycin and its binding partners in autocrine promotion of metastasis in esophageal cancer.

Rationale: Little is known about the roles of proteoglycans in esophageal cancer. This study aims to investigate the roles and mechanisms of serglycin (SRGN) proteoglycan in promoting metastasis of esophageal squamous cell carcinoma (ESCC). Methods: Reverse phase protein array analysis was used to identify activated signaling pathways in SRGN-overexpressing cells. Chemokine array was used to identify differentially secreted factors from SRGN-overexpressing cells. Binding between SRGN and potential interacting partners was evaluated using proximity ligation assay and co-immunoprecipitation. The glycosaminoglycan (GAG) chains of SRGN were characterized using fluorophore-assisted carbohydrate electrophoresis. Tissue microarray and serum samples were used to determine the correlation of SRGN expression with clinicopathological parameters and patient survival. Results: In vitro and in vivo experiments showed that SRGN promoted invasion and metastasis in ESCC via activating ERK pathway, stabilizing c-Myc and upregulating the secretion of matrix metalloproteinases. SRGN-knockdown suppressed tumorigenic hallmarks. These SRGN-elicited functions were carried out in an autocrine manner by inducing the secretion of midkine (MDK), which was further identified as a novel binding partner of SRGN for the formation of a SRGN/MDK/CD44 complex. In addition, SRGN interacted with MDK and matrix metalloproteinase 2 in ESCC via its GAG chains, which were mainly decorated with chondroitin sulfate comprising of ∆di-4S and ∆di-6S CS. Clinically, high expression of serum SRGN in serum of patients with ESCC was an independent prognostic marker for poor survival. Conclusions: This study provides the first evidence that elevated serum SRGN has prognostic significance in patients with ESCC, and sheds light on the molecular mechanism by which elevated circulating SRGN in cancer patients might promote cancer progression.

Therapeutic evaluation of palbociclib and its compatibility with other chemotherapies for primary and recurrent nasopharyngeal carcinoma.

BACKGROUND: Recent genomic analyses revealed that druggable molecule targets were only detectable in approximately 6% of patients with nasopharyngeal carcinoma (NPC). However, a dependency on dysregulated CDK4/6-cyclinD1 pathway signaling is an essential event in the pathogenesis of NPC. In this study, we aimed to evaluate the therapeutic efficacy of a specific CDK4/6 inhibitor, palbociclib, and its compatibility with other chemotherapeutic drugs for the treatment of NPC by using newly established xenograft models and cell lines derived from primary, recurrent, and metastatic NPC. METHODS: We evaluated the efficacies of palbociclib monotherapy and concurrent treatment with palbociclib and cisplatin or suberanilohydroxamic acid (SAHA) in NPC cell lines and xenograft models. RNA sequencing was then used to profile the drug response-related pathways. Palbociclib-resistant NPC cell lines were established to determine the potential use of cisplatin as a second-line treatment after the development of palbociclib resistance. We further examined the efficacy of palbociclib treatment against cisplatin-resistant NPC cells. RESULTS: In NPC cells, palbociclib monotherapy was confirmed to induce cell cycle arrest in the G1 phase in vitro. Palbociclib monotherapy also had significant inhibitory effects in all six tested NPC tumor models in vivo, as indicated by substantial reductions in the total tumor volumes and in Ki-67 proliferation marker expression. In NPC cells, concurrent palbociclib treatment mitigated the cytotoxic effect of cisplatin in vitro. Notably, concurrent treatment with palbociclib and SAHA synergistically promoted NPC cell death both in vitro and in vivo. This combination also further inhibited tumor growth by inducing autophagy-associated cell death. NPC cell lines with induced palbociclib or cisplatin resistance remained sensitive to treatment with cisplatin or palbociclib, respectively. CONCLUSIONS: Our study findings provide essential support for the use of palbociclib as an alternative therapy for NPC and increase awareness of the effective timing of palbociclib administration with other chemotherapeutic drugs. Our results provide a foundation for the design of first-in-human clinical trials of palbociclib regimens in patients with NPC.

Nondestructive quantification of single-cell nuclear and cytoplasmic mechanical properties based on large whole-cell deformation.

The mechanical properties of cell nuclei have been recognized to reflect and modulate important cell behaviors such as migration and cancer cell malignant tendency. However, these nuclear properties are difficult to characterize accurately using conventional measurement methods, which are often based on probing or deforming local sites over a nuclear region. The corresponding results are sensitive to the measurement position, and they are not decoupled from the cytoplasmic properties. Microfluidics is widely recognized as a promising technique for bioassay and phenotyping. In this report, we develop a simple and nondestructive approach for the single-cell quantification of nuclear elasticity based on microfluidics by considering different deformation levels of a live cell captured along a confining microchannel. We apply two inlet pressure levels to drive the flow of human nasopharyngeal epithelial cells (NP460) and human nasopharyngeal cancerous cells (NPC43) into the microchannels. A model considering the essential intracellular components (cytoplasm and nucleus) for describing the mechanics of a cell deforming along the confining microchannel is used to back-calculate the cytoplasmic and nuclear properties. On the other hand, we also apply a widely used chemical nucleus extraction technique to examine its possible effects (e.g., reduced nuclear modulus and reduced lamin A/C expression). To determine if the decoupled nuclear properties are representative of cancer-related attributes, we classify the NP460 and NPC43 cells using the decoupled physical properties as classification factors, resulting in an accuracy of 79.1% and a cell-type specificity exceeding 74%. It should be mentioned that the cells can be recollected at the device outlet after the nondestructive measurement. Hence, the reported cell elasticity measurement can be combined with downstream genetic and biochemical assays for general cell research and cancer diagnostic applications.

EBV-encoded miRNAs can sensitize nasopharyngeal carcinoma to chemotherapeutic drugs by targeting BRCA1.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy. The high expression of BART-miRNAs (miR-BARTs) during latent EBV infection in NPC strongly supports their pathological importance in cancer progression. Recently, we found that several BART-miRNAs work co-operatively to modulate the DNA damage response (DDR) by reducing Ataxia-telangiectasia-mutated (ATM) activity. In this study, we further investigated the role of miR-BARTs on DDR. The immunohistochemical study showed that the DNA repair gene, BRCA1, is consistently down-regulated in primary NPCs. Using computer prediction programs and a series of reporter assays, we subsequently identified the negative regulatory role of BART2-3p, BART12, BART17-5p and BART19-3p in BRCA1 expression. The ectopic expression of these four miR-BARTs suppressed endogenous BRCA1 expression in EBV-negative epithelial cell lines, whereas BRCA1 expression was enhanced by repressing endogenous miR-BARTs activities in C666-1 cells. More importantly, suppressing BRCA1 expression in nasopharyngeal epithelial cell lines using miR-BART17-5p and miR-BART19-3p mimics reduced the DNA repair capability and increased the cell sensitivity to the DNA-damaging chemotherapeutic drugs, cisplatin and doxorubicin. Our findings suggest that miR-BARTs play a novel role in DDR and may facilitate the development of effective NPC therapies.

Exosomal Delivery of AntagomiRs Targeting Viral and Cellular MicroRNAs Synergistically Inhibits Cancer Angiogenesis.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer characterized by a high degree of recurrence, angiogenesis, and metastasis. The importance of alternative pro-angiogenesis pathways including viral factors has emerged after decades of directly targeting various signaling components. Using NPC as a model, we identified an essential oncogenic pathway underlying angiogenesis regulation that involves the inhibition of a tumor suppressor, Spry3, and its downstream targets by EBV-miR-BART10-5p (BART10-5p) and hsa-miR-18a (miR-18a). Overexpression of EBV-miR-BART10-5p and hsa-miR-18a strongly promotes angiogenesis in vitro and in vivo by regulating the expression of VEGF and HIF1-α in a Spry3-dependent manner. In vitro or in vivo treatment with iRGD-tagged exosomes containing antagomiR-BART10-5p and antagomiR-18a preferentially suppressed the angiogenesis and growth of NPC. Our findings first highlight the role of EBV-miR-BART10-5p and oncogenic hsa-miR-18a in NPC angiogenesis and also shed new insights into the clinical intervention and therapeutic strategies for nasopharyngeal carcinoma and other virus-associated tumors.

Role of miR-96/EVI1/miR-449a Axis in the Nasopharyngeal Carcinoma Cell Migration and Tumor Sphere Formation.

The Wnt signaling pathway is one of the major signaling pathways used by cancer stem cells (CSC). Ecotropic Viral Integration Site 1 (EVI1) has recently been shown to regulate oncogenic development of tumor cells by interacting with multiple signaling pathways, including the Wnt signaling. In the present study, we found that the Wnt modulator ICG-001 could inhibit the expression of EVI1 in nasopharyngeal carcinoma (NPC) cells. Results from loss-of-function and gain-of-function studies revealed that EVI1 expression positively regulated both NPC cell migration and growth of CSC-enriched tumor spheres. Subsequent studies indicated ICG-001 inhibited EVI1 expression via upregulated expression of miR-96. Results from EVI1 3'UTR luciferase reporter assay confirmed that EVI1 is a direct target of miR-96. Further mechanistic studies revealed that ICG-001, overexpression of miR-96, or knockdown of EVI1 expression could restore the expression of miR-449a. The suppressive effect of miR-449a on the cell migration and tumor sphere formation was confirmed in NPC cells. Taken together, the miR-96/EVI1/miR-449a axis is a novel pathway involved in ICG-001-mediated inhibition of NPC cell migration and growth of the tumor spheres.