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EBV latent membrane proteins promote hybrid epithelial-mesenchymal and extreme mesenchymal states of nasopharyngeal carcinoma cells for tumorigenicity.

ABSTRACT: EBV-encoded LMPs are consistently detected in nasopharyngeal carcinoma (NPC). Recent evidence suggests potential roles of LMP1 and LMP2A in Epithelial-to-mesenchymal transition (EMT) process in NPC. EMT engages in the generation and maintenance of cancer stem cells (CSCs) and confers on cancer cells increased tumor-initiating and metastatic potential, and higher resistance to anticancer therapies. However, how LMP1 and LMP2A regulate the EMT process to generate cells with different EMT states and its implications for tumor progression remain unclear. Here we report that LMP1 and LMP2A promote EMT that drives NPC cells from the epithelial-like state (E) (CD104+, CD44low) to epithelial-mesenchymal hybrid (E/M) state (CD104+, CD44high). Furthermore, LMP2A possesses an additional function in stabilizing LMP1 and increasing the level of LMP1 in NPC cells. The elevated LMP1 further forces the EMT to generate extreme-mesenchymal (xM) state cells (CD104-, CD44high). To define the tumorigenic features of cancer stem cells at different states in the EMT spectrum, E, E/M and xM subpopulations were isolated and tested for tumorigenic capability in a tumor xenograft animal model. We found that the cells with E/M phenotypes possess the highest tumor initiating capacity. However, the xM subpopulation exhibits increased vasculogenic mimicry, a hallmark of metastatic cancers. Taken together, coordinated action of LMP1 and LMP2A generates an array of intermediate subpopulations in the EMT spectrum that are responsible for distinct tumorigenic features of NPC such as tumor-initiation, vasculogenesis, and metastasis.

SUBMITTER: Zhu N

PROVIDER: S-EPMC8405006 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nasopharyngeal carcinoma (NPC) is common among southern Chinese including the ethnic Cantonese population living in Hong Kong. Epstein-Barr virus (EBV) infection is detected in all undifferentiated type of NPC in this endemic region. Establishment of stable and latent EBV infection in premalignant nasopharyngeal epithelial cells is an early event in NPC development and may contribute to its pathogenesis. Immortalized primary nasopharyngeal epithelial cells represent an important tool for investigation of EBV infection and its tumorigenic potential in this special type of epithelial cells. However, the limited availability and small sizes of nasopharyngeal biopsies have seriously restricted the establishment of primary nasopharyngeal epithelial cells for immortalization. A reliable and effective method to immortalize primary nasopharyngeal epithelial cells will provide unrestricted materials for EBV infection studies. An earlier study has reported that Bmi-1 expression could immortalize primary nasopharyngeal epithelial cells. However, its efficiency and actions in immortalization have not been fully characterized. Our studies showed that Bmi-1 expression alone has limited ability to immortalize primary nasopharyngeal epithelial cells and additional events are often required for its immortalization action. We have identified some of the key events associated with the immortalization of primary nasopharyngeal epithelial cells. Efficient immortalization of nasopharyngeal epithelial cells could be reproducibly and efficiently achieved by the combined actions of Bmi-1 expression, activation of telomerase and silencing of p16 gene. Activation of MAPK signaling and gene expression downstream of Bmi-1 were detected in the immortalized nasopharyngeal epithelial cells and may play a role in immortalization. Furthermore, these newly immortalized nasopharyngeal epithelial cells are susceptible to EBV infection and supported a type II latent EBV infection program characteristic of EBV-infected nasopharyngeal carcinoma. The establishment of an efficient method to immortalize primary nasopharyngeal epithelial cells will facilitate the investigation into the role of EBV infection in pathogenesis of nasopharyngeal carcinoma.

Project description:BackgroundEpstein-Barr virus (EBV) is ubiquitously associated with nasopharyngeal carcinoma (NPC). EBV encodes two groups of microRNAs (miRNAs) which are divided into BamHI fragment H rightward open reading frame 1 (BHRF1) and BamHI-A rightward transcripts (BART) microRNAs. EBV miR-BART has been found to be involved in the development and progression of NPC. However, so far the role of EBV-miR-BART8-3p in NPC progression remains unknown. This study aimed to investigate the role of EBV-miR-BART8-3p in NPC and explore the underlying mechanisms.MethodsmiRNA expression was profiled in NPC and normal nasopharyngeal mucosal specimens using miRNA sequencing. EBV-miR-BART8-3p and RNF38 expression was quantified with qPCR assay. The migration, invasion and metastasis of NPC cells were evaluated using CCK-8, colony-forming, wound-healing, and migration and invasion assays. The expression levels of epithelial-mesenchymal transition (EMT)-related markers,metastasis-related markers and NF-κB and Erk1/2 signaling proteins were determined using Western blotting. Tumorigenic assay was performed to evaluate the pulmonary metastatic ability of NPC cells in vivo.ResultsEBV BART miRNAs were highly over-expressed and co-expressed in NPC and might be associated with deactivated immune response in NPC according to the sequencing analysis. EBV-miR-BART8-3p expression was significantly higher in human NPC specimens than in normal nasopharyngeal mucosal specimens. EBV-miR-BART8-3p was found to promote NPC migration, invasion and metastasis, drove an EMT process and upregulated expression of metastasis-related proteins expression in NPC cells. Our data showed EBV-miR-BART8-3p directly targeted RNF38 in NPC cells.ConclusionThe present study demonstrates that EBV-miR-BART8-3p plays a significant role in inducing EMT and promoting metastasis through directly targeting RNF38 in NPC cells via the activation of NF-κB and Erk1/2 signaling pathways. Our findings suggest that EBV-miR-BART8-3p is a potential therapeutic target for NPC.

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EBV latent membrane proteins promote hybrid epithelial-mesenchymal and extreme mesenchymal states of nasopharyngeal carcinoma cells for tumorigenicity.

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