Nasopharyngeal Carcinoma Model Research Articles

MEK inhibition by trametinib overcomes chemoresistance in preclinical nasopharyngeal carcinoma models.

The development of chemoresistance is the major cause of treatment failure in nasopharyngeal carcinoma (NPC). Although 'paradoxical' activation of extracellular signal-regulated kinase (ERK) has been shown to contribute resistance to anticancer treatment, the role of ERK in NPC chemoresistance has not been yet revealed. In this work, we report that trametinib, a clinically available mitogen-activated protein kinase inhibitor for melanoma treatment, overcomes NPC chemoresistance via suppressing ERK activation induced by chemotherapy. We first showed that trametinib at nanomolar concentrations was active against NPC cells and acted synergistically with cisplatin. Trametinib remarkably decreased phosphorylation of ERK and its downstream effector in NPC cells. We next showed that cisplatin treatment stimulates ERK signaling, and furthermore that this can be abolished by trametinib. We finally generated cisplatin-resistant NPC models and demonstrated that trametinib was effective in inhibiting cisplatin-resistant NPC growth, colony formation and survival via suppressing ERK signaling in vitro and in vivo. Our work demonstrates the potential of trametinib in overcoming chemoresistance in preclinical NPC models and provides evidence of initializing clinical trials of using trametinib for NPC treatment.

Abstract 948: Preclinical evaluation of VEGF Ang2 bispecific nanobody BI836880 in patient-derived xenograft models of nasopharyngeal carcinoma

Abstract BI836880 is a humanized bispecific nanobody directed against angiopoietin-2 (Ang2) and vascular endothelial growth factor (VEGF)-derived peptides, with selective and potent antiangiogenic and antineoplastic activities. It comprises of blocking domains that bind to Ang2 and VEGF and prevents Ang2- and VEGF-mediated signalling; thus, inhibits both angiogenesis and tumour cell proliferation. Tumor angiogenesis contributes to progression of nasopharyngeal cancer (NPC) and VEGF/receptor inhibitors agents have activity in NPC patients. In this study, the preclinical activity of a novel VEGF/Ang-2 bi-specific nanobody, BI836880, was evaluated in two NPC-PDX models - Xeno-2117 and Xeno-666. The effect of BI836880 on tumor growth was compared with the control, bevacizumab - an anti-VEGF antibody which has been evaluated in NPC patients. Tumor-bearing mice were randomized into three groups: vehicle control, 15mg/kg bevacizumab (b.i.w) and 15mg/kg BI836880 (b.i.w.). Drug was administered by intraperitoneal injection and the treatment duration was four weeks. Tumor sizes and body weights were monitored by caliper and balance, respectively. The results showed that BI836880 was very well tolerated in mice, with no signs of stress and loss of weight observed during the treatment. At day 14, the mean tumor volumes of BI836880, bevacizumab vs vehicle control was 264.5±36.0, 290.0±32.4 vs 697.38±88.4 mm3 in Xeno-666 (p<0.001, BI836880 vs control), and 293.5±39.3, 319.6±37.6 vs 1016.8±107.6 mm3 in Xeno-2117 (p<0.0001, BI836880 vs control), respectively. Angiogenesis marker CD34 was analyzed by IHC staining and the microvessel densities were calculated by Image J. The results showed that BI836880 could significantly reduce the numbers of microvessels to a comparable extent as bevacizumab in NPC model. In addition, BI836880 had increased the tumor internal necrotic area by 118.7% compared to vehicle control in Xeno-2117 (p=0.0148), but the effect was insignificant in Xeno-666 (p=0.5467). In conclusion, BI836880 can inhibit NPC growth to an extent that is comparable to bevacizumab, further investigation in combination with cytotoxic or immunotherapeutic agents are warranted. Citation Format: Eric Chi Wong, Gigi Lam, Connie Wong, Rachel C. Lam, Kwok Wai Lo, Edwin Pun Hui, Anthony Tak Chan, Brigette B. Ma. Preclinical evaluation of VEGF Ang2 bispecific nanobody BI836880 in patient-derived xenograft models of nasopharyngeal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 948.

Apatinib for the treatment of metastatic or locoregionally recurrent nasopharyngeal carcinoma after failure of chemotherapy: A multicenter, single‐arm, prospective phase 2 study

The authors aimed to investigate the efficacy and safety of apatinib in patients with metastatic or locoregionally recurrent nasopharyngeal carcinoma (NPC). A multicenter, single-arm, prospective phase 2 study was conducted on patients (18-70 years of age) with metastatic or recurrent NPC who had failed chemotherapy. Patients with recurrent disease involving vascular structure invasion were excluded. All enrolled patients received apatinib (500 mg daily) in continuous 4-week cycles until disease progression or development of unacceptable toxicity. The primary end point of this study was objective response rate (ORR), and the secondary end points were progression-free survival (PFS), overall survival (OS), and toxicity. This study was registered with ClinicalTrials.gov (NCT03130270). Between January 2017 and June 2018, 33 patients were enrolled. At the end of the data collection (May 20, 2020), the 33 patients had completed a total of 261.2 cycles of apatinib. Although 12 patients achieved a partial response, no patient achieved a complete response; thus, the ORR in the 33 patients was 36.4% (95% CI, 19.0%-53.7%). At the end of follow-up (median, 30 months; 95% CI, 24.9-35.1), median OS and median PFS were 16 months (95% CI, 14.6-17.4 months) and 5.0 months (95% CI, 3.6-6.4 months), respectively. The most common adverse events (grade 1/2) were hand-foot syndrome (18 [54.5%]), hypertension (14 [42.4%]), oral ulcer (8 [24.2%]), and proteinuria (4 [12.1%]). Two patients (1 with diabetes and 1 with hypertension) developed cerebral infarction. Grade 3/4 toxicities were uncommon. Apatinib shows promising activity, with manageable toxicities, in patients with metastatic or locoregionally recurrent NPC. Further evaluation of apatinib in large-scale studies is warranted. Clinical studies on vascular endothelial growth factor receptor (VEGFR)-targeted therapy for recurrent or metastatic nasopharyngeal carcinoma (NPC) are limited. A recent preclinical study that evaluated apatinib in models of NPC showed a high objective response rate and a favorable safety profile. Our data further confirmed good efficacy in patients with lung metastasis. Further studies of the efficacy and safety of apatinib combined with immune checkpoint inhibitors or chemotherapy in NPC is warranted.

Computer-aided diagnosis and regional segmentation of nasopharyngeal carcinoma based on multi-modality medical images.

Nasopharyngeal carcinoma (NPC) is a category of tumors with high incidence in head-and-neck (H&N) body region, and the diagnosis and treatment planning are usually conducted by radiologists manually, which is tedious, time-consuming and unrepeatable. In this paper, we integrated different stages of this process and proposed a computer-aided framework to realize automatic detection, tumor region and sub-region segmentation, and visualization of NPC, which are usually investigated separately in literatures. Multi-modality images are utilized in the framework. Firstly, NPC is detected by a convolutional neural network (CNN) on computed tomography (CT) scans. Then, NPC area is segmented from magnetic resonance imaging (MRI) images by using a multi-modality MRI fusion network. Thirdly, NPC sub-regions with different metabolic activities are divided on CT images of the same patient via an adaptive threshold algorithm. Finally, 3D surface model of NPC is generated for observing its shape, size, and location in the head region. The proposed method is compared with other algorithms by evaluation on the volumes and shapes of detected NPCs. Experiments are conducted on CT images of 130 NPC patients and 102 subjects without NPC and MRI images of 149 NPC patients, among which 52 subjects are overlapped with both CT and MRI images. The reference for evaluation is generated by three experienced radiologists. The results demonstrated that our utilized models outperform other strategies with detection accuracy 0.882 and Dice similarity coefficient 0.719 for NPC segmentation. Sub-region division and the 3D visualized models show great acceptability in clinical usage. The remarkable performance indicated the potential of our framework in alleviating workload of radiologist. Furthermore, the combined usage of multi-modality images is able to generate reliable segmentations of NPC area and sub-regions, which provide evidence to judge the heterogeneity among patients and guide the dose painting for radiation therapy.

Nuclear magnetic resonance spectrum aided diagnosis based on DNN neural network

The nuclear magnetic resonance spectrum contains a variety of effective information, and most of the current clinical medicine uses nuclear magnetic resonance images as a diagnostic basis, but the spectral information is still not effectively explored. In order to improve the diagnostic results of nuclear magnetic resonance spectrum, this study uses DNN neural network as a technical support to extract effective information of nuclear magnetic resonance spectrum. Simultaneously, in order to improve the ability to describe the local features of the image, the traditional Crow algorithm is improved, and a similar target localization algorithm based on F-CroW is proposed. In addition, starting from the animal model of nasopharyngeal carcinoma and the serum and urine samples of patients with clinical nasopharyngeal carcinoma, this study designed a comparative study to study the performance of the proposed algorithm. According to the research and analysis, the DNN neural network proposed in this study has certain effects in the nuclear magnetic resonance spectrum analysis, which can be applied to clinical practice.

Identification of miPEP133 as a novel tumor-suppressor microprotein encoded by miR-34a pri-miRNA

BackgroundVery few proteins encoded by the presumed non-coding RNA transcripts have been identified. Their cellular functions remain largely unknown. This study identifies the tumor-suppressor function of a novel microprotein encoded by the precursor of miR-34a. It consists of 133 amino acid residues, thereby named as miPEP133 (pri-microRNA encoded peptide 133).MethodsWe overexpressed miPEP133 in nasopharyngeal carcinoma (NPC), ovarian cancer and cervical cancer cell lines to determine its effects on cell growth, apoptosis, migration, or invasion. Its impact on tumor growth was evaluated in a xenograft NPC model. Its prognostic value was analyzed using NPC clinical samples. We also conducted western blot, immunoprecipitation, mass spectrometry, confocal microscopy and flow cytometry to determine the underlying mechanisms of miPEP133 function and regulation.ResultsmiPEP133 was expressed in normal human colon, stomach, ovary, uterus and pharynx. It was downregulated in cancer cell lines and tumors. miPEP133 overexpression induced apoptosis in cancer cells and inhibited their migration and invasion. miPEP133 inhibited tumor growth in vivo. Low miPEP133 expression was an unfavorable prognostic marker associated with advanced metastatic NPC. Wild-type p53 but not mutant p53 induced miPEP133 expression. miPEP133 enhanced p53 transcriptional activation and miR-34a expression. miPEP133 localized in the mitochondria to interact with mitochondrial heat shock protein 70kD (HSPA9) and prevent HSPA9 from interacting with its binding partners, leading to the decrease of mitochondrial membrane potential and mitochondrial mass.ConclusionmiPEP133 is a tumor suppressor localized in the mitochondria. It is a potential prognostic marker and therapeutic target for multiple types of cancers.

Abstract 2923: Targeting the polycomb repressive complex-2 related proteins in nasopharyngeal carcinoma

Abstract Aberrant hypermethylation of tumor suppressor genes is common in nasopharyngeal carcinoma (NPC), which is also enriched in denovo methylation at PCR2-related gene loci. PRC2 complex has three core subunits: embryonic ectoderm development (EED), enhancer of Zeste 1/2 (EZH1/2) and suppressor of Zeste 12 (SUZ12), which can mediate gene silencing. This study investigated the pharmacological effect of targeting EED and EZH2 in NPC cell lines (C666-1, C17C, NPC43 and HK1) and an immortalized normal nasopharyngeal epithelial cell line (NP69), either alone or concomitantly with other drugs. The relevance and prognostic significance of EED, SUZ12, EZH2 and H3K27me3 expression were determined in 100 NPC samples. EZH2 and EED was strongly expressed in EBV-positive NPC cell lines (C666-1, C17C and NPC43), but weakly in EBV negative cell line HK1 and NP69. The effect of EED inhibitor (EED226) and EZH2 inhibitor (EPZ-6438) on cell growth was tested in C666-1, NPC43 and HK1. Treatment with EED226 reduced the level of H3K27me3, with only minimal/ modest inhibition on NPC cell growth at 72hr. Drug combinations of PRC2-targeting agents with other epigenetic modulators or chemotherapy drugs were evaluated at, and near their IC50 concentrations in C666-1 and NPC43. Three combinations indicated synergistic effect: (i) EPZ-6438 and HDAC inhibitor (TSA) (synergy score, SS = 12.636); (ii) EED226 and gemcitabine (SS = 10.285); (iii) DNMT inhibitor (AZA) and EED226 (SS = 7.336). RNA-seq analysis were done after treatment with 5M of EED226 for 3, 7 and 14 days. GO enrichment analysis, KEGG pathway enrichment analysis and Artificial Neural Network (ANN) were used for bioinformatics analysis. EED226 at low dose mainly affected the cell-adherence related genes at 3-7 days after treatment, and MHC protein complex and cell cycle related genes were significantly changed after longer drug exposure (7-14 days) in NPC cells. ANN predicted a combination therapy of EED226 and LEE011 (CDK4/6 inhibitor) and we demonstrated a synergistic inhibitory effect on cell growth when EED226 was combined with LEE011 in C666-1 and HK1 cells at 72hr, with the respective combination index (CI) of ED50 < 0.7. In conclusion, EED226 can significantly down- regulate the level of H3K27me3 at low dose, but with minimal growth inhibitory effect as monotherapy. EED and EZH2 inhibitor have synergistic effect when combined with chemotherapy and other epigenetic modulators and CDK4/6 inhibitors in vitro. Validation in vivo model of NPC is warranted. Data on the prognostic of PRC2-related protein significance in advanced NPC will be presented. Acknowledgement of grant: Direct grant for research, The Chinese University of Hong Kong (#2018.012). Citation Format: Junyu ZHU, Lili Li, Connie W. Hui, Joanna H. Tong, Raymond Chan, Chi Hang Wong, Qiyong Ai, Edwin P. Hui, Anthony T. Chan, Kwok Wai Lo, Qian Tao, Brigette B. Ma. Targeting the polycomb repressive complex-2 related proteins in nasopharyngeal carcinoma [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 2923.

Abstract 5060: Establishment and characterization of humanized mouse NPC-PDX model for testing immunotherapy

Abstract Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) is common in Southern China and South-East Asia. Immune checkpoint blockade (ICB) is being studied in NPC and initial monotherapy ICB studies show early promise. Currently, lack of representative NPC models limit the preclinical studies to evaluate the efficacy and safety of novel ICB and combination regimens. Hence, there is a need to develop and characterize a mouse model with human immune system for testing immunotherapy. Here, we engrafted NPC-PDX in immunodeficient NSG mice, with a successful growth rate of 16.7% (6 out of 36), and humanized mouse NPC-PDX model was established subsequently. Of note, EBV latent membrane protein-1 was retained in the NPC in both NSG and humanized mice as revealed by IHC staining. There was an increase in tumor-infiltrating CD4+ T cells and a decrease in CD8+ cytotoxic T cells in humanized mice, when compared to the immune cells profile in circulation. Intriguingly, the expression levels of various exhaustion markers, namely PD-1, CTLA4 and TIM-3 were upregulated, particularly in the tumor-infiltrating T cells. Therefore, the use of ICB, either alone or in combination, may restore the function of anti-tumor immune effector cells. Taken together, we have established a humanized mouse NPC-PDX model, which plausibly provides a robust platform to test for the efficacy and safety of ICB and combination therapy regimens. Citation Format: Wai Nam LIU, Shin Yie FONG, Wilson Wei Sheng TAN, Sue Yee TAN, Min LIU, Narayanan Gopalakrishna IYER, Darren Wan-Teck LIM, Qingfeng CHEN. Establishment and characterization of humanized mouse NPC-PDX model for testing immunotherapy [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 5060.

Targeting Epstein-Barr Virus in Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is consistently associated with Epstein-Barr virus (EBV) infection in regions in which it is endemic, including Southern China and Southeast Asia. The high mortality rates of NPC patients with advanced and recurrent disease highlight the urgent need for effective treatments. While recent genomic studies have revealed few druggable targets, the unique interaction between the EBV infection and host cells in NPC strongly implies that targeting EBV may be an efficient approach to cure this virus-associated cancer. Key features of EBV-associated NPC are the persistence of an episomal EBV genome and the requirement for multiple viral latent gene products to enable malignant transformation. Many translational studies have been conducted to exploit these unique features to develop pharmaceutical agents and therapeutic strategies that target EBV latent proteins and induce lytic reactivation in NPC. In particular, inhibitors of the EBV latent protein EBNA1 have been intensively explored, because of this protein's essential roles in maintaining EBV latency and viral genome replication in NPC cells. In addition, recent advances in chemical bioengineering are driving the development of therapeutic agents targeting the critical functional regions of EBNA1. Promising therapeutic effects of the resulting EBNA1-specific inhibitors have been shown in EBV-positive NPC tumors. The efficacy of multiple classes of EBV lytic inducers for NPC cytolytic therapy has also been long investigated. However, the lytic-induction efficiency of these compounds varies among different EBV-positive NPC models in a cell-context-dependent manner. In each tumor, NPC cells can evolve and acquire somatic changes to maintain EBV latency during cancer progression. Unfortunately, the poor understanding of the cellular mechanisms regulating EBV latency-to-lytic switching in NPC cells limits the clinical application of EBV cytolytic treatment. In this review, we discuss the potential approaches for improvement of the above-mentioned EBV-targeting strategies.

Establishment and Characterization of Humanized Mouse NPC-PDX Model for Testing Immunotherapy.

Immune checkpoint blockade (ICB) monotherapy shows early promise for the treatment of nasopharyngeal carcinoma (NPC) in patients. Nevertheless, limited representative NPC models hamper preclinical studies to evaluate the efficacy of novel ICB and combination regimens. In the present study, we engrafted NPC biopsies in non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain-null (NSG) mice and established humanized mouse NPC-patient-derived xenograft (NPC-PDX) model successfully. Epstein–Barr virus was detected in the NPC in both NSG and humanized mice as revealed by Epstein–Barr virus-encoded small RNA (EBER) in situ hybridization (ISH) and immunohistochemical (IHC) staining. In the NPC-bearing humanized mice, the percentage of tumor-infiltrating CD8+ cytotoxic T cells was lowered, and the T cells expressed higher levels of various inhibitory receptors, such as programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) than those in blood. The mice were then treated with nivolumab and ipilimumab, and the anti-tumor efficacy of combination immunotherapy was examined. In line with paired clinical data, the NPC-PDX did not respond to the treatment in terms of tumor burden, whilst an immunomodulatory response was elicited in the humanized mice. From our results, human proinflammatory cytokines, such as interferon-gamma (IFN-γ) and interleukin-6 (IL-6) were significantly upregulated in plasma. After treatment, there was a decrease in CD4/CD8 ratio in the NPC-PDX, which also simulated the modulation of intratumoral CD4/CD8 profile from the corresponding donor. In addition, tumor-infiltrating T cells were re-activated and secreted more IFN-γ towards ex vivo stimulation, suggesting that other factors, including soluble mediators and metabolic milieu in tumor microenvironment may counteract the effect of ICB treatment and contribute to the tumor progression in the mice. Taken together, we have established and characterized a novel humanized mouse NPC-PDX model, which plausibly serves as a robust platform to test for the efficacy of immunotherapy and may predict clinical outcomes in NPC patients.

Difference of PDT induced apoptotic gene expression in 2D and 3D nasopharyngeal carcinoma cell models

Abstract Background Nasopharyngeal Carcinoma (NPC) is one of the top 10 cancers in Hong Kong. Photodynamic Therapy (PDT) is an alternative treatment approach to NPC. We previously reported that PDT induced both apoptosis and necrosis in 2D and 3D NPC models but in different manners, yet the underline biological mechanism is still unknown. In this pilot study we measured 3 apoptotic related gene expression, including c-Myc, p53 and Notch-1 genes using 2D and 3D EBV+ c666-1 cell models at pre- and post- FosPeg (FP) PDT treatment. Methods Total 5 x 105 cells were used for 2D and 3D multicellular layer cell models. Cells were activated with laser at 20J/cm2 after 24 hours FP incubation. Total RNA were obtained using high pure RNA isolation kit (Roche) and were converted to cDNA. 20ng cDNA were used for qPCR (Light Cycler 480). Results Results revealed that 2D and 3D models expressed different mRNA levels of c-Myc, p53 and Notch-1 gene, in both control cells and PDT treated cells. Results indicated that there were 0.3 to 1.2 fold difference in mRNA expression between 2D and 3D cell models among the tested apoptotic genes. 2D model was found more susceptible to PDT inducing mRNA expression than 3D model. The difference could be explained by: 1) light and drug delivery variation; 2) intracellular oxygen difference; and 3) cell to cell communication difference in 2D and 3D cell models. Conclusion 2D and 3D cell models behave differently at both molecular and cellular levels. These differences may due to different PDT responses in 2D and 3D models. 3D cell models were found to be more suitable for PDT pharmacokinetic studies. Further investigation is needed on PDT mediated molecular changes (protein expression) in 3D models. Acknowledgement The work described in this paper was fully supported by a grant from the Research Grants Council of the Hong Kong Special Administrative Region, China (project no.:2015-00-74-RGC150201).

Translational genomics of nasopharyngeal cancer

Nasopharyngeal carcinoma (NPC), also named the Cantonese cancer, is a unique cancer with strong etiological association with infection of the Epstein-Barr virus (EBV). With particularly high prevalence in Southeast Asia, the involvement of EBV and genetic aberrations contributive to NPC tumorigenesis have remained unclear for decades. Recently, genomic analysis of NPC has defined it as a genetically homogeneous cancer, driven largely by NF-κB signaling caused by either somatic aberrations of NF-κB negative regulators or by overexpression of the latent membrane protein 1 (LMP1), an EBV viral oncoprotein. This represents a landmark finding of the NPC genome. Exome and RNA sequencing data from new EBV-positive NPC models also highlight the importance of PI3K pathway aberrations in NPC. We also realize for the first time that NPC mutational burden, mutational signatures, MAPK/PI3K aberrations, and MHC Class I gene aberrations, are prognostic for patient outcome. Together, these multiple genomic discoveries begin to shape the focus of NPC therapy development. Given the challenge of NF-κB targeting in human cancers, more innovative drug discovery approaches should be explored to target the unique atypical NF-κB activation feature of NPC. Our next decade of NPC research should focus on further identification of the -omic landscapes of recurrent and metastatic NPC, development of gene-based precision medicines, as well as large-scale drug screening with the newly developed and well-characterized EBV-positive NPC models. Focused preclinical and clinical investigations on these major directions may identify new and effective targeting strategies to further improve survival of NPC patients.

Patient-derived tumor models for human nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common head and neck tumors in Southern China. At present, the interaction of genetic susceptibility, Epstein-Barr virus (EBV) infection and environmental factors has been considered to be the main cause of NPC. However, the detailed molecular mechanisms of tumorigenesis and tumor metastasis have not been fully understood. The effective therapeutic drugs targeting NPC are still being developed and discovered. NPC cell lines and normal nasopharyngeal epithelial cell lines were frequently used by researchers, but not represent the complex situation in vivo. Establishing an ideal animal model of NPC is one of the keys to solving the above problems. Here, we introduce the development of in vitro and in vivo models of NPC.

Establishment and characterization of new tumor xenografts and cancer cell lines from EBV-positive nasopharyngeal carcinoma

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.

High NEK2 confers to poor prognosis and contributes to cisplatin-based chemotherapy resistance in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor in southern China and Southeast Asia, but the molecular mechanism of its pathogenesis is poorly understood. Our previous work demonstrated that NEK2 is overexpressed in multiple cancers. However, how NEK2 involves in NPC development remains to be elucidated. In this study, we firstly identified NEK2, located at +1q32‐q33, a late event in NPC pathogenesis, overexpressed in the stage III‐IV and paired sequential recurrent patients with NPC by immunohistochemistry. Furthermore, Kaplan‐Meier analysis indicated high NEK2 conferred an inferior overall survival in NPC. In addition, cisplatin experiments with cell counting kit‐8, colony formation, and a xenograft mice model of NPC demonstrated that NEK2 contributed to proliferation and cisplatin resistance in vitro and in vivo. On the contrary, downregulation of NEK2 by short hairpin RNA inhibited NPC cell growth and increased the sensitivity of cisplatin treatment in vitro. Thus, increased expression of NEK2 protein could not be predicted for poor survival but used as a novel biomarker for recurrence of NPC. Targeting NEK2 has the potential to eradicate the cisplatin‐based chemotherapy resistant NPC cells.

Preclinical evaluation of ribociclib and its synergistic effect in combination with alpelisib in non-keratinizing nasopharyngeal carcinoma

Ribociclib is a specific cyclin dependent kinase (Cdk) 4/6 inhibitor that induces G1 arrest by blocking the formation of cyclin D1-Cdk4/6 complex and inhibiting retinoblastoma (RB) phosphorylation. Cyclin D1 is overexpressed in over 90% of nasopharyngeal carcinoma (NPC) and CCND1 gene activation plays a critical role in NPC pathogenesis. This study evaluated the preclinical activities of ribociclib in NPC cell lines and patient derived xenograft (PDX) models. Over 95% cell growth inhibition was observed at 96 hours after ribociclib treatment. (IC50 concentrations: HK1 = 1.42 ± 0.23 µM; HK1-LMP1 = 2.18 ± 0.70 µM and C666-1 = 8.26 ± 0.92 µM). HK1 and C666-1 cells were chosen for analysis of ribociclib on kinase signaling, apoptosis and cell cycle. Treatment with ribociclib for 48 hours consistently showed a dose-dependent reduction in phosphorylated and total RB expression and G1 cycle arrest was only observed. Combining ribociclib with the alpha-specific PI3K inhibitor alpelisib showed a synergistic effect in two NPC PDX models in nude mice. The co-treatment induced a significant reduction in tumor volume in both xeno-666 and xeno-2117 compared with ribociclib treatment alone and control (p < 0.01). In summary, ribociclib is active in NPC models and the effect on growth inhibition was augmented when combined with alpelisib. This study supports the clinical evaluation of ribociclib in NPC.

KAI1/CD82 Genetically Engineered Endothelial Progenitor Cells Inhibit Metastasis of Human Nasopharyngeal Carcinoma in a Mouse Model.

BackgroundEndothelial progenitor cells (EPCs) are regarded as promising targeted vectors for delivering therapeutic genes or agents in cancer therapy. The purpose of this study was to investigate the role of intravenously administered KAI1/CD82 genetically transduced EPCs in the tumorigenesis and metastasis of nasopharyngeal carcinoma (NPC).Material/MethodsEPCs were isolated from human umbilical cord blood, expanded in culture, and stably transduced with lentiviral vectors expressing KAI1/CD82. The KAI1/CD82 EPCs were injected intravenously into nude mice bearing human NPC xenografts. Tumor growth and the incidence of liver and lung metastases were observed. Expression of KAI1/CD82 was determined by immunofluorescent staining.ResultsThe NPC model was successfully established. Tumor growth was not suppressed when mice were injected with KAI1/CD82 EPCs (KAI1/CD82 EPCs group) compared with when non-transduced EPCs was present (EPCs group) or the control (1.485±0.234, 1.388±0.204, and 1.487±0.223g, respectively; P>0.05). However, the incidence of lung metastasis was significantly reduced in the KAI1/CD82+ EPCs group compared with the EPCs group and the control group (10%, 55% and 45%, respectively; P=0.005), and there was a significant decrease in the number of metastatic foci on the lung surface (17.50±3.54, 34.27±5.35, and 38.44±9.63 respectively; P=0.007). Moreover, KAI1/CD82 was expressed in lung metastatic foci of the KAI1/CD82 EPCs group, but not in the EPCs group and control group.ConclusionsEPCs can be used as a delivery vehicle for suppressor genes KAI1/CD82 to NPC, and the migration of KAI1/CD82 genetically engineered EPCs can inhibit NPC lung metastasis in a mouse model.

Influence ofPOLGon Radiosensitivity of Nasopharyngeal Carcinoma Cells

There is a high incidence of nasopharyngeal carcinoma (NPC), malignant head and neck tumors, in southern China. Radioresistance is the main cause affecting the efficacy of NPC treatments. The POLG gene particularly plays an important role in radiation-induced damage repair. In this study, the authors established RNAi CNE-1 and CNE-2 knockdown in two NPC cell lines to observe whether this gene affects the radiosensitivity of NPC cells. Four short hairpin RNA (shRNA) expression plasmids targeting POLG gene were constructed and transfected into the NPC cell lines CNE-1 and CNE-2. Screening was performed to evaluate the stable expression of cloned cells, which were named CNE-1/POLG-shRNA1, CNE-1/POLG-shRNA2, CNE-2/POLG-shRNA1, and CNE-2/POLG-shRNA2. The negative controls CNE-1/Neg-shRNA and CNE-2/Neg-shRNA were additionally used. The MTT method, flow cytometry, clone formation analysis, cell migration, and other experimental methods were employed to verify changes in the radiosensitivity of the NPC cells. Fluorescent quantitative PCR and Western blot confirmed the downregulation of the PLOG gene through diminished PLOG messenger RNA and protein levels. Consequently, the authors report the stable knockdown of the POLG gene in an NPC model. Dose-dependent radiation exposure of POLG inhibited NPC cell growth and increased apoptosis compared with control cells (p < 0.01), as demonstrated through colony formation assay and flow cytometry. Functional assays indicated that knockdown of the POLG in CNE-1 and CNE-2 cells remarkably reduced cell viability and proliferation. Specifically, POLG knockdown led to G1 phase arrest and apoptosis. Overall, the authors conclude that POLG downregulation alters the radiosensitivity of NPC cells, indicating that the gene is likely involved in conferring the radiation response of the cells. In addition, findings in this study suggest a novel role for POLG as a potential predictive marker for NPC radiotherapy efficiency. POLG gene can be used as a potential clinical target to effectively improve the radiosensitivity of NPC.

Baicalein inhibits growth of Epstein-Barr virus-positive nasopharyngeal carcinoma by repressing the activity of EBNA1 Q-promoter

Epstein-Barr virus (EBV) can establish a life-long latent infection in the host and is associated with various human malignancies, including nasopharyngeal carcinoma (NPC), the most common cancer originated from nasopharynx. EBV nuclear antigen 1 (EBNA1) is the only viral protein absolutely demanded for segregation, replication, transcription and maintenance of EBV viral genome in host cells. Baicalein, a bioactive flavonoid compound purified from the root of Scutellariae baicaleinsis, displays anti-inflammatory, immunosuppressive, and anti-tumor properties. In this study, the therapeutic effects and functional mechanism of baicalein on EBV-positive human NPC were determined. Cell Counting Kit-8 assays and cell formation colony were performed to investigate that baicalein can suppress proliferation of EBV-infected human NPC cells. Flow cytometric and hoechst 33258 staining results indicated that baicalein induced cell cycle arrest and apoptosis. Western blotting results demonstrated that baicalein down-regulates EBNA1 expression but not reduces the stability and half-life of EBNA1 in EBV-infected NPC cells. Additionally, the mRNA level of EBNA1 was examined by real time-PCR, the activity of EBNA1 Q promoter (Qp) was determined by dual luciferase reporter assay. Considering that transcription factor specificity protein 1 (Sp1) can maintain EBNA1 Qp active. Further analyses also elucidated that baicalein inhibits the expression of Sp1 while knock-down Sp1 by specific shRNAs decreases the expression and transcription levels of EBNA1. Therefore, the results suggested that baicalein may decrease EBNA1 expression level in EBV-positive NPC cells via inhibiting the activity of EBNA1 Q-promoter while over-expression of EBNA1 attenuate the inhibitory effect of baicalein. Finally, it was found that baicalein may strongly reduce growth of tumor in the mouse xenograft model of EBV-positive NPC. These results indicated that baicalein inhibits growth of EBV-positive NPC by repressing the activity of EBNA1 Q-promoter. Baicalein may be used as a therapeutic agent to treat EBV-positive NPC.

Long noncoding RNA NEAT1 promotes nasopharyngeal carcinoma progression through regulation of miR-124/NF-κB pathway.

Nasopharyngeal carcinoma (NPC) is one of the most common malignancies and seriously endangers people’s health. Recently, long noncoding RNA (lncRNA) NEAT1 has been determined as an oncogenic gene in a variety of cancers. However, the effect of NEAT1 in NPC and its underlying mechanism have not been well elaborated. In this study, the data showed that NEAT1 was upregulated and miR-124 was downregulated in NPC tissues and cells. Loss-of-function revealed that NEAT1 knockdown inhibited proliferation and promoted apoptosis of NPC cells while gain-of-function revealed that upregulated NEAT1 showed an opposite effect. Moreover, NEAT1 was demonstrated to suppress miR-124 expression by direct interaction in NPC cells. Additionally, miR-124 reversed NEAT1-mediated pro-proliferation and anti-apoptosis effect. Furthermore, miR-124 regulated NPC cell proliferation and apoptosis via NF-κB signal pathway. Mouse models of NPC confirmed that NEAT1 overexpression facilitated tumor growth by modulating miR-124 in vivo. Taken together, this study indicated that upregulated NEAT1 promoted the tumorigenesis and progression of NPC through regulating miR-124/NF-κB signaling pathway, suggesting an attractive therapy target for NPC patients.