Nasopharyngeal Carcinoma Tumor Cells Research Articles

Abstract 465: Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors

Abstract Most humans infected with Epstein-Barr virus (EBV) remain asymptomatic throughout the whole life. However, EBV is linked to several human cancers, including Burkitt's lymphoma, Hodgkin's disease, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC), and gastric carcinoma. Among these EBV-associated tumors, EBV genome is present in 100% of NPC tumor cells. In NPC cells, EBV establishes a latent infection, expressing limited number of viral proteins but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, also known as BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs includes BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). Although various functions of BART miRNAs have been identified, the role of EBV lnc-BARTs remains largely unknown. In this study, we provide evidence demonstrating that lnc-BARTs functions as a regulatory long non-coding RNA, referred to as lnc-BARTs. These lnc-BARTs play keys role in modulating the core networks that maintains EBV latency and promotes NPC oncogenesis through epigenetic mechanisms. Our study found that the apoptosis rate of NPC cells is significantly increased following the knockdown of EBV lnc-BARTs, indicating their anti-apoptotic role in EBV-associated tumors. Mass spectrometry analysis identified several host factors, including transcriptional regulators BRD4 and SC35’ interact with EBV lnc-BARTs in NPC cells. RNA-FISH assays showed lnc-BARTs co-localized with BRD4 and SC35 complexes in nuclear s peckles and such interaction were disrupted by JQ1, a BRD4 competitive inhibitor. Binding of lnc-BARTs to BRD4 RNA was confirmed using immunoprecipitation and pulldown assays. ATAC sequencing and transcriptome analyses showed that BART knockdown in EBV-harboring NPC cell line C666-1 led to reduced chromatin accessibility, downregulation of oncogenes, and decreased EBV episome replication and maintenance. Quantitative qPCR and DNA FISH analyses of cell samples treated with shRNA targeting lnc-BARTs or BRD4 reduced EBV copy numbers and EBNA1 RNA expression levels in EBV-infected NPC cells. Finally, ChIP sequencing analysis revealed that lnc-BARTs is critical for BRD4 binding to the oriP region of the EBV genome. These findings support a mechanism by which EBV lnc-BARTs epigenetically regulate host gene expression through functional interactions with elongation regulatory machinery and contribute to the maintenance of EBV latent genome by interaction with BRD4 complex. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Honglin Chen. Effect of Epstein-Barr virus lnc-BARTs on EBV genome maintenance in EBV-associated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 465.

Soluble CD27 as a predictive biomarker for intra-tumoral CD70/CD27 interaction in nasopharyngeal carcinoma.

In CD70-expressing tumors, the interaction of CD70 on tumor cells with its lymphocyte receptor, CD27, is thought to play a role in immunosuppression in the tumor microenvironment and elevated serum levels of soluble CD27 (sCD27). Previous studies showed that CD70 is expressed in nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-related malignancy. However, the association between intratumoral CD70/CD27 expression and serum levels of sCD27 in NPC remains unclear. In the present study, we show that CD70 is primarily expressed by tumor cells in NPC and that CD27-positive lymphocytes infiltrate around tumor cells. NPC patients with CD27-positive lymphocytes had significantly better prognosis than patients lacking these cells. In addition, high CD70 expression by tumor cells tended to be correlated with shorter survival in NPC patients with CD27-positive lymphocytes. Serum sCD27 levels were significantly increased in patients with NPC and provided good diagnostic accuracy for discriminating patients from healthy individuals. The concentration of serum sCD27 in patients with CD70-positive NPC with CD27-positive lymphocytes was significantly higher than in patients with tumors negative for CD70 and/or CD27, indicating that the intratumoral CD70/CD27 interaction boosts the release of sCD27. Furthermore, positive expression of CD70 by NPC cells was significantly correlated with EBV infection. Our results suggest that CD70/CD27-targeted immunotherapies may be promising treatment options and that sCD27 may become an essential tool for evaluating the applicability of these therapies by predicting the intratumoral CD70/CD27 interaction in NPC.

Single-cell analysis reveals the multiple patterns of immune escape in the nasopharyngeal carcinoma microenvironment.

Single-cell transcriptomics has revolutionised our understanding of the cellular composition of the tumour microenvironment (TME) in nasopharyngeal carcinoma (NPC). Despite this progress, a key limitation of this technique has been its inability to capture epithelial/tumour cells, which has hindered further investigation of tumour heterogeneity and immune escape in NPC. In this study, we aimed to address these limitations by analysing the transcriptomics and spatial characteristics of NPC tumour cells at single-cell resolution using scRNA/snRNA-seq and imaging mass cytometry techniques. Our findings demonstrate multiple patterns of immune escape mechanisms in NPC, including the loss of major histocompatibility complex (MHC) molecules in malignant cells, induction of epithelial-mesenchymal transition in fibroblast-like malignant cells and the use of hyperplastic cells in tumour nests to protect tumour cells from immune infiltration. Additionally, we identified, for the first time, a CD8+ natural killer (NK) cell cluster that is specific to the NPC TME. These findings provide new insights into the complexity of NPC immune landscape and may lead to novel therapeutic strategies for this disease.

Therapeutic hyperthermia regulates complement C3 activation and suppresses tumor development through HSPA5/NFκB/CD55 pathway in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is endemic in Southern China and Southeast Asia. Hyperthermia is widely used in combination with chemotherapy and radiotherapy to enhance therapeutic efficacy in NPC treatment, but the underlying anti-tumor mechanisms of hyperthermia remain unclear. Complement C3 has been reported to participate in the activation of immune system in the tumor microenvironment, leading to tumor growth inhibition. In this study, we aimed to explore the effect and mechanisms of hyperthermia and investigate the functional role of complement C3 in NPC hyperthermia therapy (HT). The serum levels of complement C3 before and after hyperthermia therapy in patients with NPC were analyzed. NPC cell lines SUNE1 and HONE1 were used for in vitro experiment to evaluate the function of complement C3 and HT on cell proliferation and apoptosis. SUNE1 xenograft mouse model was established and tumor-bearing mice were treated in water bath at a constant temperature of 43°C. Tumor samples were collected at different time points to verify the expression of complement C3 by immunohistochemical staining and western blot. The differential expressed genes after hyperthermia were analyzed by using RNA sequencing. We found that complement could enhance hyperthermia effect on suppressing proliferation and promoting apoptosis of tumor cells in NPC. Hyperthermia decreased the mRNA expression of complement C3 in tumor cells, but promoted the aggregation and activation circulating C3 in NPC tumor tissue. By using in vitro hyperthermia-treated NPC cell lines and SUNE1 xenograft tumor-bearing mice, we found that the expression of heat shock protein 5 (HSPA5) was significantly upregulated. Knockdown of HSPA5 abrogated the anti-tumor effect of hyperthermia. Moreover, we demonstrated that hyperthermia downregulated CD55 expression via HSPA5/NFκB (P65) signaling and activated complement cascade. Our findings suggest that therapeutic hyperthermia regulates complement C3 activation and suppresses tumor development via HSPA5/NFκB/CD55 pathway in NPC.

Abstract 3749: Oncogenic role of Epstein-Barr virus lnc-BARTs in EBV associated tumors

Abstract While majority of humans infected with Epstein-Barr virus (EBV) is asymptomatic for whole life, EBV is found to associate with several human cancers, including Burkitt’s lymphoma, Hodgkin’s diseases, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC) and gastric carcinoma. Among these EBV associated tumors, EBV genome is found in 100% of NPC tumor cells. EBV establishes a latent infection in NPC cells, expressing few viral proteins, but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, designated BamHI-A rightward transcripts (BARTs). The family of EBV BART RNAs comprises BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). While versatile functions of BART miRNAs have been revealed, little is known about the role of EBV lnc-BARTs. Here, we provided evidence to show that BARTs mRNA which derived from multiple exons of BART function as regulatory long non-coding RNA, namely lnc-BARTs, in modulating the core network for maintaining EBV latency and promoting NPC oncogenesis through epigenetic mechanisms. Our results showed that the apoptosis rate of NPC cell lines with knockdown of EBV lnc-BARTs was significantly increased, suggesting that lnc-BARTs have an anti-apoptotic effect in EBV associated tumors. MS analysis was performed to identify host factors which interact with EBV lnc-BARTs in NPC cells. Lnc-BARTs were shown to directly interact with several transcriptional regulators, including BRD4 and SC35. Notably, RNA-FISH assays demonstrated that lnc-BARTs co-localize with BRD4 and SC35 complexes in nuclear speckles, with these complexes being disrupted by treatment with JQ1, a BRD4 competitive inhibitor. RNA immunoprecipitation and RNA pulldown assays confirmed that lnc-BARTs bind to BRD4. ATAC sequencing analysis and transcriptome analysis further demonstrated that knockdown of BARTs in the EBV-harboring NPC cell line, C666-1, resulted in globally reduced chromatin accessibility downregulation of oncogenes like MYC and BCL2. In addition, ChIP sequencing analysis revealed that lnc-BARTs interfere PolII phosphorylation on promoters of genes. Our findings suggest that EBV lnc-BARTs are involved in epigenetic modulation of host gene expression through functional interaction with elongation regulatory machinery to maintain EBV latency and drive tumorigenesis in NPC. Citation Format: LIU JIAYAN, Bobo Wing-Yee Mok, Songtao He, Honglin Chen. Oncogenic role of Epstein-Barr virus lnc-BARTs in EBV associated tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3749.

A glutathione-sensitive cationic polymer delivery of CRISPR-Cas9 RNA plasmid for targeting nasopharyngeal carcinoma gene therapy

CRISPR-Cas9 technology has been proven to be the most straightforward and accurate tool for gene therapy, but some limitations, such as the inefficient transfection or inability to precisely target, prevent the gene therapy from achieving the desired therapeutic effect. To overcome these, a kind of glutathione-sensitive cationic vectors, hyperbranched polyamide amine (HPAA) was designed for Delivery of CRISPR-Cas9 RNA plasmid, and the cyclic RGD (Arg-Gly-Asp) was conjugated for the targeted treatment of nasopharyngeal carcinoma (NPC). Disulfide bonds in HPAA segments can specifically respond to the high glutathione concentration in the tumor microenvironment. Meanwhile, RGD could especially interact to the integrin αvβ3 receptors which are highly expressed on the surface of NPC tumor cells. The results showed that more HPAA-RGD/SGK3-gRNA complexes could be uptaken by NPC HNE-1 cells after RGD was conjugated, and then the plasmid could be accumulated in the NPC tumor as well, which may assure the satisfied NPC therapy effect in vivo. In transfection assays, this complex showed the acceptable gene transfection efficiency in vitro and the obvious tumor inhibition effect in vivo, suggested a potential application in gene therapy to NPC.

Antipsychotic drug trifluoperazine as a potential therapeutic agent against nasopharyngeal carcinoma.

Trifluoperazine (TFP) is a typical antipsychotic primarily used to treat schizophrenia. In this study, we aimed to evaluate whether TFP can be used as a therapeutic agent against nasopharyngeal carcinoma (NPC) and identify its underlying molecular mechanisms. We used NPC-TW01, TW03, TW04, and BM to assess the anticancer effects of TFP by using cytotoxicity, wound healing, colony formation, and cell invasion assays. An in vivo animal study was conducted. RNA sequencing combined with Ingenuity Pathways Analysis was performed to identify the mechanism by which TFP influences NPC cells. Our data revealed that TFP decreased NPC cell viability in a dose-dependent manner. The invasion and migration of NPC tumor cells were inhibited by TFP. An in vivo study also demonstrated the anticancer effects of TFP. RNA sequencing revealed several anticancer molecular mechanisms following TFP administration. The antipsychotic drug TFP could be a potential therapeutic regimen for NPC treatment.

Lenvatinib for effectively treating antiangiogenic drug-resistant nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) clinical trials show that antiangiogenic drugs (AADs) fail to achieve the expected efficacy, and combining AAD with chemoradiotherapy does not show superiority over chemoradiotherapy alone. Accumulating evidence suggests the intrinsic AAD resistance in NPC patients with poorly understood molecular mechanisms. Here, we describe NPC-specific FGF-2 expression-triggered, VEGF-independent angiogenesis as a mechanism of AAD resistance. Angiogenic factors screening between AAD-sensitive cancer type and AAD-resistant NPC showed high FGF-2 expression in NPC in both xenograft models and clinical samples. Mechanistically, the FGF-2-FGFR1-MYC axis drove endothelial cell survival and proliferation as an alternative to VEGF-VEGFR2-MYC signaling. Genetic knockdown of FGF-2 in NPC tumor cells reduced tumor angiogenesis, enhanced AAD sensitivity, and reduced pulmonary metastasis. Moreover, lenvatinib, an FDA recently approved multi-kinase inhibitor targeting both VEGFR2 and FGFR1, effectively inhibits the tumor vasculature, and exhibited robust anti-tumor effects in NPC-bearing nude mice and humanized mice compared with an agent equivalent to bevacizumab. These findings provide mechanistic insights on FGF-2 signaling in the modulation of VEGF pathway activation in the NPC microenvironment and propose an effective NPC-targeted therapy by using a clinically available drug.

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

BackgroundRadiotherapy 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...

Rapid nasopharyngeal brush-smear cytology with Epstein-Barr virus DNA load and viral marker analysis for detection of nasopharyngeal carcinoma

Background: Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Indonesia, usually identified at late stage. Epstein-Barr virus (EBV) gene products are present in all tumor cells. Early-stage NPC diagnosis is clinically important but difficult due to non- specific symptoms. In patients suspected of having NPC, nasopharyngeal (NP) brush-cytology may provide a simple non-invasive procedure for rapid on-site detection of NPC tumour cells that can be combined with EBV markers. Methods: Patients (N=83) suspected of having NPC underwent nasoendoscopy-guided NP brushing before a biopsy from clinically suspicious areas. The loaded brush was first smeared onto glass slides that were directly immersed in ethanol, followed by Papanicolaou (PAP) staining and on-site interpretation of tumour cell presence. Thereafter, slides were used for EBER RNA in situ hybridization (EBER-ISH) and LMP-1 immunocytochemistry (ICC). The brush left-over was used for EBV-DNA load measurement using real-time PCR. Biopsy specimens were taken from the same NP site and processed for routine pathology and EBER-ISH on tissue sections. Result: Morphological detection of NPC tumour cells on PAP-stained brush-smears gave a sensitivity and specificity of 87% and 100%, respectively. EBER-RISH and LMP1 ICC combined showed 100% positivity for NPC cases. Expression of LMP-1 was detected in 77.8% of cases. Median EBV-DNA load in left-over brush material from NPC cases was 5.06 × 106 copies/brush (range 777 to 242,400,000 copies/brush), whereas median value in non-NPC cases was 4.4 x 103 copies/brush (range 484 - 297,200; P < 0.001). Conclusion: Minimal invasive NP-brush cytology with conventional PAP staining allows rapid, simple and cheap “on site” detection of NPC tumour cells in suspected NP lesions. The method can be modified for direct demonstration of EBV involvement using EBER-ISH and LMP1-ICC or EBV-DNA load measurement. This NP brush procedure may be useful for primary screening in patients considered at risk for NPC.

Long-chain non-coding RNA LOC554202 promotes proliferation, migration, and invasion of nasopharyngeal carcinoma cells by binding to microRNA-31 expression and regulating RhoA expression.

Long-chain non-coding LOC554202, as a host gene for microRNA-31, has been shown to play a crucial role in a variety of diseases, especially tumors. However, its biological function in nasopharyngeal carcinoma (NPC) has not been reported. The expression levels of LOC554202 and microRNA-31 in NPC tumor tissue samples and cell lines were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). The impacts of LOC554202 and microRNA-31 on the biological functions of NPC cells were examined by Cell Counting Kit-8 (CCK-8) and transwell assays. In addition, the modulation of LOC554202 on the expressions of microRNA-31 and RhoA was further confirmed by qRT-PCR and Western blot analysis. The data of this study indicated that LOC554202 expression in NPC tissues and cell lines was remarkably upregulated, while microRNA-31 level showed an opposite tendency. Increasing LOC554202 expression remarkably enhanced the growth and metastasis of NPC cells, which was inhibited by overexpression of microRNA-31. Overexpression of LOC554202 downregulated microRNA-31 expression but upregulated that of RhoA, which may be a potential mechanism for the implication of LOC554202 in NPC. As a host gene of microRNA-31, LOC554202 enhances RhoA expression and thus promotes the proliferative capacity and invasiveness of NPC cells.

Epstein-Barr Virus Infection of Pseudostratified Nasopharyngeal Epithelium Disrupts Epithelial Integrity.

Simple SummaryNasopharyngeal carcinoma is associated with Epstein-Barr virus (EBV) infection and originates junction of the oropharynx and nasal cavity, where stratified squamous epithelium and respiratory epithelium are the lining. To elucidate the mechanisms by which EBV transforms the nasopharyngeal epithelium, a pseudostratified multiple-layer model with cilia forming on the apical surface by air-liquid interface (ALI) culture of primary nasopharyngeal epithelial cells was established. We showed: (1) ALI cultures formed stratified epithelia and maintained the diversity of cells found in the airway epithelium, such as ciliated, muco-secretory, and basal cells. (2) Polarized stratified epithelium was more susceptible to EBV infection than monolayer cells. (3) EBV infection in ALI cultures was verified by showing EBV-encoded RNA expressions. (4) EBV infection disrupted the integrity of the epithelium. Thus, our model can be used not only to examine the pathogenesis of pre-neoplastic EBV-infected cells, but also to develop anti-EBV therapy or early stage NPC treatment.Epstein–Barr virus (EBV) is a human oncogenic virus that causes several types of tumor, such as Burkitt’s lymphoma and nasopharyngeal carcinoma (NPC). NPC tumor cells are clonal expansions of latently EBV-infected epithelial cells. However, the mechanisms by which EBV transforms the nasopharyngeal epithelium is hampered, because of the lack of good in vitro model to pursue oncogenic process. Our primary nasopharyngeal epithelial cell cultures developed pseudostratified epithelium at the air-liquid interface, which was susceptible to EBV infection. Using the highly sensitive RNA in situ hybridization technique, we detected viral infection in diverse cell types, including ciliated cells, goblet cells, and basal cells. EBV-encoded small RNA-positive cells were more frequently detected in the suprabasal layer than in the basal layer. We established the most physiologically relevant EBV infection model of nasopharyngeal epithelial cells. This model will advance our understanding of EBV pathogenesis in the development of NPC.

Abstract 272: Oncogenic role of epstein-barr virus lnc-BARTs in nasopharyngeal carcinoma cells

Abstract Epstein-Barr virus (EBV) is associated with several human cancers, including Burkitt's lymphoma, Hodgkin's diseases, post-transplant lymphoproliferative disorder (PTLD), nasopharyngeal carcinoma (NPC) and gastric carcinoma. Among these EBV associated tumors, EBV genome is found in 100% of NPC tumor cells. EBV establishes a latent infection in NPC cells, expressing few viral proteins, but elevated levels of non-coding RNAs transcribed from the BamHI-A region of the EBV genome, designated BamHI-A rightward transcripts (BARTs). The family of BART RNAs comprises BART-microRNAs (miRNAs) and long non-coding RNAs (lnc-BARTs). While versatile functions of BART miRNAs continue to be revealed, little is known about EBV lnc-BARTs. Here, we provide evidence to show that lnc-BARTs derived from multiple exons of BART RNA function as nuclear long non-coding RNAs in modulating host gene expression in NPC cells. We found that lnc-BARTs upregulate transcription of SEPT9, matrix metalloproteinase (MMP) genes and IKZF3 (Aiolos) and accelerate N-cadherin cleavage. As a member of the Septin family, SEPT9 (cytoskeletal interacting protein, Septin 9) is involved in cytokinesis and cell cycle control. Aiolos can negatively regulate expression of p66Shc, which is associated with the oxidative stress response and apoptosis, and our data also showed that knockout of IKZF3 in NPC cells enhanced transcription of p66Shc protein. Our results indicated that the apoptosis rate of NPC cell lines with knockdown of lnc-BARTs was significantly increased, suggesting that lnc-BARTs have an anti-apoptotic effect in EBV associated tumors. To understand the molecular basis of gene regulation by EBV BART-lncRNAs, ChIRP-MS analysis was performed: lnc-BARTs were shown to directly interact with several transcriptional regulators, including BRD4 and KDM1B. Notably, RNA FISH assays demonstrated that lnc-BARTs co-localize with BRD4 and P-TEFb complexes in nuclear speckles, with these complexes being disrupted by treatment with JQ1, a BRD4 competitive inhibitor. RNA immunoprecipitation and RNA pulldown assays confirmed that lnc-BARTs bind directly to BRD4. In addition, transcriptome analysis revealed that knockdown of BARTs in the EBV-harboring NPC cell line, C666-1, resulted in downregulation of MYC and BCL2, genes downstream of BRD4. BRD4-ChIP and ATAC sequencing analysis further demonstrated that knockdown of lnc-BARTs reduced both BRD4-mediated epigenetic modulation of host gene expression and chromatin accessibility. We suggest that EBV lnc-BARTs are involved in epigenetic modulation of host gene expression through functional interaction with BRD4 regulatory machinery to maintain EBV latency in NPC cells, and that lnc-BARTs modulate host gene expression to drive tumorigenesis in NPC. Citation Format: Jiayan Liu, Bobo Wing-Yee Mok, Songtao He, Honglin Chen. Oncogenic role of epstein-barr virus lnc-BARTs in nasopharyngeal carcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 272.

Skp2-mediated ubiquitination and mitochondrial localization of Akt drive tumor growth and chemoresistance to cisplatin.

The E3 ligase S-phase kinase-associated protein 2(Skp2) is overexpressed in human cancers and correlated with poor prognosis, but its contributions to tumorigenesis and chemoresistance in nasopharyngeal carcinoma (NPC) are not evident. Herein we show that Skp2 is highly expressed in NPC tumor tissues and cell lines. Knockdown of Skp2 suppresses tumor cell growth, colony formation, glycolysis, and in vivo tumor growth. Skp2 promotes Akt K63-mediated ubiquitination and activation, which is required for EGF-induced Akt mitochondrial localization. Importantly, K63-linked ubiquitination enhances the interaction between Akt and HK2 and eventually increases HK2 phosphorylation on Thr473 and mitochondrial localization. Overexpression of Skp2 impaired the intrinsic apoptotic pathway and confers cisplatin resistance. Moreover, ectopic expression of Myr-Akt1 or phosphomimetic HK2-T473D rescued cisplatin-induced tumor suppression in Skp2 knockdown stable cells. Also, depletion of Akt ubiquitination enhances the antitumor efficacy of cisplatin in vitro and in vivo. Finally, we demonstrated that Skp2 is positively correlated with p-Akt and HK2 in NPC tumor tissues. This study highlights the clinical value of Skp2 targeting in NPC treatment.

Abstract 403: COX-2 expression on circulating tumor cells in nasopharyngeal carcinoma predicts a worse survival: A prospective analysis

Abstract COX-2 expression on circulating tumor cells in nasopharyngeal carcinoma predicts a worse survival: A prospective analysis Yong-Jiang Li1, Xiao-Qi Xie2, Feng Wang1* 1Department of Oncology, West China Hospital, Sichuan University, Chengdu, PR China 2Department of Critical Care Medicine, West China Hospital, Sichuan University, Chengdu, PR China *Corresponding author Short title: COX-2 Expression on CTCs in NPC patients Abstract Background and purpose: The prognostic significance of circulating tumor cells (CTCs) in head and neck cancer is still under debate, as only a few studies have been reported and limited conclusions are reached. Besides, CTCs count alone was utilized as an indicator in the previous researches. As a form of ‘liquid biopsy’, the further identification of genetic or phenotypic biomarkers on CTCs could possibly provide further clinical significance. Materials and methods: The prospective study enrolled 131 patients with nasopharyngeal carcinoma (NPC). CTCs were isolated at baseline and at the end of concurrent chemoradiotherapy, and cyclooxygenase-2 (COX-2) expression status of CTCs was detected by RNA-in situ hybridization (ISH) method. Results were correlated with patient’s clinicopathological parameters and treatment outcomes. Univariate and multivariate survival analysis were performed to determine the prognostic significance. Results: COX-2 expression was found in 87/131 (66.4%) patients at baseline and 53/115 (46.1%) patients at the end of concurrent chemoradiotherapy. Patients with post-therapeutic COX-2 expression had significantly poorer treatment response (P=0.011) and higher risk of tumor relapse (P=0.026) and metastasis (P=0.007). Besides, multivariate analysis revealed that post-therapeutic COX-2 expression on CTCs remained an independent prognostic indicator for poorer progression-free survival (HR 2.17, P=0.019) and overall survival (HR 2.41, P=0.024). Conclusion: The study demonstrated that post-therapeutic COX-2 expression on CTCs was a novel and promising prognostic indicator for NPC patients. Future studies are needed to validate our findings and further clarify the value of integrating the indicator with current clinical strategies in improving survival of NPC patients. Note: This abstract was not presented at the meeting. Citation Format: Yongjiang Li, Xiaoqi Xie, Feng Wang. COX-2 expression on circulating tumor cells in nasopharyngeal carcinoma predicts a worse survival: A prospective analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 403.

Vesicle-bound EBV-BART13-3p miRNA in circulation distinguishes nasopharyngeal from other head and neck cancer and asymptomatic EBV-infections.

Cell‐free microRNA (miRNA) in biofluids released by tumors in either protein or vesicle‐bound form, represent promising minimally‐invasive cancer biomarkers. However, a highly abundant non‐tumor background in human plasma and serum complicates the discovery and detection of tumor‐selective circulating miRNAs. We performed small RNA sequencing on serum and plasma RNA from Nasopharyngeal Carcinoma (NPC) patients. Collectively, Epstein Barr virus‐encoded miRNAs, more so than endogenous miRNAs, signify presence of NPC. However, RNAseq‐based EBV miRNA profiles differ between NPC patients, suggesting inter‐tumor heterogeneity or divergent secretory characteristics. We determined with sensitive qRT‐PCR assays that EBV miRNAs BART7‐3p, BART9‐3p and BART13‐3p are actively secreted by C666.1 NPC cells bound to extracellular vesicles (EVs) and soluble ribonucleoprotein complexes. Importantly, these miRNAs are expressed in all primary NPC tumor biopsies and readily detected in nasopharyngeal brushings from both early and late‐stage NPC patients. Increased levels of BART7‐3p, BART9‐3p and particularly BART13‐3p, distinguish NPC patient sera from healthy controls. Receiver operating characteristic curve analysis using sera from endemic NPC patients, other head and neck cancers and individuals with asymptomatic EBV‐infections reveals a superior diagnostic performance of EBV miRNAs over anti‐EBNA1 IgA serology and EBV‐DNA load (AUC 0.87–0.96 vs 0.86 and 0.66 respectively). The high specificity of circulating EBV‐BART13‐3p (97%) for NPC detection is in agreement with active secretion from NPC tumor cells. We conclude EV‐bound BART13‐3p in circulation is a promising, NPC‐selective, biomarker that should be considered as part of a screening strategy to identify NPC in endemic regions.

M6A-mediated ZNF750 repression facilitates nasopharyngeal carcinoma progression

Nasopharyngeal carcinoma (NPC) progression is regulated by genetic, epigenetic, and epitranscript modulation. As one of the epitranscript modifications, the role of N6-Methyladenosine (m6A) has not been elucidated in NPC. In the present study, we found that the poorly methylated gene ZNF750 (encoding zinc finger protein 750) was downregulated in NPC tumor tissues and cell lines. Ectopic expression of ZNF750 blocked NPC growth in vitro and in vivo. Further studies revealed that m6A modifications maintained the low expression level of ZNF750 in NPC. Chromatin immunoprecipitation sequencing identified that ZNF750 directly regulated FGF14 (encoding fibroblast growth factor 14), ablation of which reversed ZNF750’s tumor repressor effect. Moreover, the ZNF750-FGF14 signaling axis inhibited NPC growth by promoting cell apoptosis. These findings uncovered the critical role of m6A in NPC, and stressed the regulatory function of the ZNF750-FGF14 signaling axis in modulating NPC progression, which provides theoretical guidance for the clinical treatment of NPC.

MicroRNA 141 represses nasopharyngeal carcinoma growth through inhibiting BMI1

Distant metastasis is the primary cause of mortality in patients with nasopharyngeal carcinoma (NPC). Unveiling the mechanism of metastasis will aid in shedding light on the clinical therapeutic strategies in NPC treatment. The present study revealed that the expression of microRNA 141 (miR-141) was downregulated in NPC tumor cells, particularly in metastatic ones. Ectopic expression of miR-141 blocked the proliferative and invasive ability of the tumor cells in vitro, and inhibited NPC tumor growth in vivo. Mechanistic studies identified that BMI1 served as the direct target of miR-141, and overexpression of BMI1 reversed the tumor repressor effect of miR-141. Prognostic analysis revealed that this miR-141/BMI1 signaling axis correlated with the clinical stage of patients with NPC. The study of miR-141 provided novel insight into the mechanism of NPC progression, which was correlated with the stage and metastatic state of patients. This miR-141/BMI1 axis may serve as a novel pharmacological target in NPC treatment.

Macrocyclic lactones inhibit nasopharyngeal carcinoma cells proliferation through PAK1 inhibition and reduce in vivo tumor growth.

PurposeThe Epstein–Barr virus (EBV)-associated cancer nasopharyngeal carcinoma (NPC) is rare in Europe and North America but is a real public health problem in some regions of the world, such as southern Asia, North Africa, and for Inuit populations. Due to the anatomy and location of the nasopharynx, surgery is rarely used to treat primary NPC cancers. Treatment by radiotherapy, combined or not with chemotherapy, are efficient for primary tumors but often do not protect against fatal relapses or metastases.MethodsSearch for new therapeutic molecules through high content screening lead to the identification of Ivermectin (IVM) as a promising drug. IVM is a US Food and Drug Administration-approved macrocyclic lactone widely used as anthelmintic and insecticidal agent that has also shown protective effects against cancers.ResultsWe show here that IVM has cytotoxic activity in vitro against NPC cells, in which it reduces MAPKs pathway activation through the inhibition PAK-1 activity. Moreover, all macrocyclic lactones tested and a PAK1 inhibitor are cytotoxic in vitro for EBV-positive and EBV-negative NPC tumor cells. We have also shown that IVM intraperitoneal repeated injections, at US Food and Drug Administration-approved doses, have no significant toxicity and decrease NPC subcutaneous tumors development in nude mice.ConclusionMacrocyclic lactones appear as promising molecules against NPC targeting PAK-1 with no detectable adverse effect.

Highlights of This Issue

The receptor tyrosine kinase AXL is aberrantly expressed in many tumors, is a potent driver of invasive cell motility and has become increasingly recognized as a broadly relevant mechanism in cancer drug resistance. Here, it is reported that exposure of cancer cells to phosphatidylserine (PS)-expressing apoptotic bodies enhances migration of tumor cells via a PS-AXL signaling axis. These findings suggest that anti-cancer therapies that induce fractional cell killing contribute to the motility of surviving cells in AXL-expressing tumors, hence generating potentially problematic invasive and metastatic behavior.Nicotinamide phosphoribosyltransferase (NAMPT) and nicotinic acid phosphoribosyltransferase (NAPRT) are rate-limiting enzymes in the nicotinamide adenine dinucleotide (NAD+) synthesis pathway. In this study, in vitro experiments reveal that chondrosarcoma cells exhibit a dose-dependent decrease in cell viability, 3D collagen invasion and colony formation upon treatment with NAMPT inhibitors. Strikingly, higher methylation of the NAPRT promoter was observed in high-grade versus low-grade chondrosarcomas. Therefore, chondrosarcoma patients, especially those of high histological grade with lower expression and hypermethylation of NAPRT, may benefit clinically from inhibition of the NAD synthesis pathway.Few actionable oncogenic driver mutations have been identified in nasopharyngeal carcinoma (NPC). However, a marked lymphocytic infiltrate and association with viral infection make immunotherapy an intriguing treatment option. Zhang and colleagues perform the first integrated analysis of gene expression, mutation, copy number, and clinical data in NPC, identifying gene expression subtypes characterized by distinct immune and Epstein-Barr virus (EBV) associated gene expression patterns. Prognostic associations with stromal tumor-infiltrating lymphocytes and a proliferation signature were also identified. This study is an important step in understanding how NPC tumor cells and EBV influence the tumor microenvironment (TME), and may help guide immunotherapy strategies in this disease.TWIST1 promotes tumorigenesis through induction of EMT, invasion and metastasis, as well as suppression of oncogene-induced senescence and apoptosis. Additionally, TWIST1 expression is required for oncogene-driven lung cancer, including tumors driven by mutant KRAS. Using a chemical-bioinformatic approach, Yochum and colleagues identified harmine as a first-in-class TWIST1 inhibitor with marked anti-tumor activity in oncogene-driven NSCLC including EGFR mutant, KRAS mutant and MET altered NSCLC. Targeting TWIST1 with a small-molecule therapeutic such as harmine and its potential analogues/derivatives, represents a novel treatment strategy in oncogene-driven lung cancer and other solid malignancies in which TWIST1 promotes tumorigenesis.