Clinical Nasopharyngeal Carcinoma Research Articles

Abstract 2553: Integration of genomic studies and the patient-derived xenograft model in nasopharyngeal carcinoma precision medicine research

Abstract Patient-derived xenograft (PDX) tumor model has become a new approach to identify druggable tumor mutations, and to screen and evaluate personalized anti-cancer drugs. Here, we established five nasopharyngeal carcinoma (NPC) PDXs in mouse model and performed whole-exome sequencing (WES) and copy number variations (CNVs) analyses. Relatively high incident rate of CNV aberration in cell cycle pathway was detected. Among these five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Selected inhibitors were tested for their anti-tumor effects in two NPC-PDX mouse models. Palbociclib, a cyclin-dependent kinase inhibitor, revealed efficient anti-tumor effects by inducing G1 arrest in the PDX tumor according to RNA sequencing. In this study, cyclin D1 overexpression was also observed in >90% clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. One NPC patient with liver metastatic who was prescribed with Palbociclib had stable disease and a drop in Epstein Barr virus (EBV) DNA titer. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients’ plasma and could serve as a screening test to select potential CDK4/6 inhibitors treatment candidates. Taken together, our findings indicate that integrated information from sequencing-based genomic studies and tumor transcriptomes before and after drug treatment in NPC-PDX models provide novel guidelines for personalized precision treatments. Citation Format: Cheng-Lung Hsu, Hsin-Pai Li. Integration of genomic studies and the patient-derived xenograft model in nasopharyngeal carcinoma precision medicine research [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 2553.

Targeting Exosomal EBV-LMP1 Transfer and miR-203 Expression via the NF-κB Pathway: The Therapeutic Role of Aspirin in NPC

Nasopharyngeal carcinoma (NPC) is an invasive head-and-neck tumor with Epstein-Barr virus (EBV) as an important etiological cause. The EBV oncoprotein Latent membrane protein 1 (LMP1) can be trafficked into exosomes with unclear roles, and this trafficking is a potential problem in NPC control. MicroRNA-203 (miR-203) was found by us to be downregulated by LMP1, and it functions as a tumor suppressor in NPC. In this study, aspirin reversed the epithelial-mesenchymal transition (EMT) by promoting miR-203 expression in cells, and, remarkably, it repressed exosomal LMP1 (exo-LMP1) secretion from EBV-positive cells. Nuclear factor κB (NF-κB) activation was required for the exo-LMP1 production. The exo-LMP1 uptake influenced the EMT potential of EBV-negative recipient NPC cells. The exo-LMP1 level was upregulated in clinical NPC plasma samples. Aspirin treatment observably inhibited NPC lung metastasis in nude mice. The study revealed that aspirin is a promising drug for NPC therapy via its targeting of exo-LMP1 transfer and the regulatory effect of LMP1 on miR-203 expression. EBV can regulate its own tumorigenesis via the LMP1/NF-κB/exo-LMP1 axis, opening a new avenue for understanding the pathogenesis of this tumor virus. Our study also provides a rationale for the use of exo-LMP1 or exosomal miR-203 (exo-miR203) in EBV-targeted therapy by aspirin in invasive NPC.

Reducing NETO2 expression prevents human nasopharyngeal carcinoma (NPC) progression by suppressing metastasis and inducing apoptosis

Nasopharyngeal carcinoma (NPC), the most common cancer in head and neck regions, is a serious health problem worldwide. Neuropilin and tolloid-like 2 (NETO2), a member of the subfamily of CUB domain and LDLa-containing proteins, has been suggested to be involved in tumor progression. Nevertheless, little is known about the function and molecular mechanism of NETO2 in NPC progression. In the study, NETO2 was found to be significantly up-regulated in clinical tissues and NPC cell lines. NETO2 expression was positively correlated with tumor size. NETO2 knockdown inhibited cell proliferation, migration and invasion in NPC cell lines. Significantly, NETO2 knockdown promoted the radiotherapy in vitro, as evidenced by the further reduced cell proliferation and metastasis in NPC cells using 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT), colony formation and transwell analysis. In addition, NETO2 inhibition markedly induced apoptosis in NPC cells through activating Caspase-3 signaling. Also, the knockdown of NETO2 obviously promoted the efficacy of radiotherapy in apoptosis induction, along with higher expression of cleaved Caspase-3. NETO2 knockdown-triggered apoptosis in NPC cells were considerably diminished by Caspase-3 inactivation, demonstrating the essential role of Caspase-3 in NETO2-regulated NPC development. Moreover, in vivo experiments suggested that NETO2 knockdown promoted radiation-induced tumor growth suppression in the absence of significant side effects. Collectively, reducing NETO2 expression might elevate the efficiency of radiotherapy in NPC patients.

Epstein-Barr virus-encoded LMP1 regulated Pim1 kinase expression promotes nasopharyngeal carcinoma cells proliferation.

BackgroundEpstein–Barr virus-encoded LMP1 plays a critical role in the carcinogenesis of nasopharyngeal carcinoma (NPC), but the mechanism remains elusive. We aimed to analyze the expression and clinical pathological significance of provirus integration site for Moloney murine leukemia virus 1 (Pim1) in clinical NPC, and to elucidate the effect of LMP1 on Pim1 expression and its mechanism.MethodsImmunohistochemical staining was used to detect the expression of Pim1 in clinical NPC tissues and control nasopharyngeal chronic inflammation (NPI) tissues, and the correlation between Pim1 and clinical parameters of NPC patients was analyzed. The LMP1 stable expression cell line CNE1-LMP1-OV was constructed through infecting the well-differentiated nasopharyngeal carcinoma cells CNE1 with LMP1 overexpressing lentivirus. Then the in vivo experiments were conducted.ResultsAmong 89 NPC patients, 48 cases (53.93%) were positive for Pim1, while only one case was Pim1 positive in 15 NPI controls (6.67%). Pim1 expression was not correlated with gender, age, smoking status and clinical classification of NPC patients, but positively correlated with T, N and M classification. CNE1-LMP1-OV cell line was successfully established, which displayed a higher cell proliferation ability and Pim1 expression. NF-κB inhibitor PDTC, PKC inhibitor GF109203X and STAT3 inhibitor Stattic significantly attenuated LMP1-induced Pim1 expression, and while AP-1 inhibitor SR11302 showed no inhibitory effect. Interestingly, Pim1 inhibitor quercetagetin significantly inhibited the proliferation of CNE1-LMP1-OV cells.ConclusionLMP1 mediates Pim1 expression through NF-κB, PKC and STAT3 signaling, which promotes the proliferation of NPC cells and participate in the clinical progression of NPC.

ATR activated by EB virus facilitates chemotherapy resistance to cisplatin or 5-fluorouracil in human nasopharyngeal carcinoma.

PurposeEpstein–Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and increases the chemotherapy resistance of tumor cells. Although the mechanism by which EBV manipulates ataxia telangiectasia mutation (ATM)-mediated DNA damage response in NPC has been extensively studied, the relationship between ATR (ATM and Rad-3 related) and EBV infection is largely unexplored, and also the role of ATR in chemotherapy resistance in EBV-positive NPC has not been specifically reported.Materials and methodsLevels of γ-H2AX, latent membrane protein 1 (LMP1), and EBV-encoded RNA in clinical NPC and nasopharyngeal inflammation (NPI) specimens were examined using immunohistochemistry and in situ hybridization. The effects of EBV infection, chemotherapy drugs cisplatin (CDDP) and 5-fluorouracil (5-FU) treatment, and ATR silencing were assessed in NPC cells in vitro using immunofluorescence, Western blot, and flow cytometry.ResultsA notable increase of γ-H2AX expression was examined in the EBV-positive NPC clinical specimens. Additionally, we observed that the phosphorylation of ATR/checkpoint kinase 1 (CHK1) pathway protein was gradually activated along with the duration of EBV exposure in NPC cell lines, which was obviously inhibited after ATR depletion. Moreover, EBV infection promoted the resistance of NPC cells to CDDP and 5-FU, whereas the chemosensitivity of cells was significantly enhanced following ATR knockdown. Furthermore, ATR depletion caused both S-phase cell arrest and apoptosis, enhanced p53 phosphorylation, and impaired the formation of Rad51.ConclusionOur data suggest that EBV activation of ATR-mediated DNA damage response might result in chemotherapy resistance to CDDP and 5-FU in NPC. Accordingly, ATR knockdown may serve as an effective treatment strategy for chemotherapy-resistant, EBV-positive NPC.

Retraction: MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance By Targeting A20/NF-κB Signaling Pathway.

Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment, but little is known about how miRNA regulates this phenomenon. In this study, we investigated the function and mechanism of miR-125b in NPC radioresistance, one of upregulated miRNAs in the radioresistant NPC cells identified by our previous microarray analysis. We observed that miR-125b was frequently upregulated in the radioresistant NPCs, and its increment was significantly correlated with NPC radioresistance, and was an independent predictor for poor patient survival. In vitro radioresponse assays showed that miR-125b inhibitor decreased, whereas miR-125b mimic increased NPC cell radioresistance. In a mouse model, therapeutic administration of miR-125b antagomir dramatically sensitized NPC xenografts to irradiation. Mechanistically, we confirmed that A20 was a direct target of miR-125b and found that miR-125b regulated NPC cell radioresponse by targeting A20/NF-κB signaling. With a combination of loss-of-function and gain-of-function approaches, we further showed that A20 overexpression decreased while A20 knockdown increased NPC cell radioresistance both in vitro and in vivo Moreover, A20 was significantly downregulated while p-p65 (RelA) significantly upregulated in the radioresistant NPCs relative to radiosensitive NPCs, and miR-125b expression level was negatively associated with A20 expression level, whereas positively associated with p-p65 (RelA) level. Our data demonstrate that miR-125b and A20 are critical regulators of NPC radioresponse, and high miR-125b expression enhances NPC radioresistance through targeting A20 and then activating the NF-κB signaling pathway, highlighting the therapeutic potential of the miR-125b/A20/NF-κB axis in clinical NPC radiosensitization. Mol Cancer Ther; 16(10); 2094-106. ©2017 AACR.

Integrated genomic analyses in PDX model reveal a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for nasopharyngeal carcinoma

BackgroundPatient-derived xenograft (PDX) tumor model has become a new approach in identifying druggable tumor mutations, screening and evaluating personalized cancer drugs based on the mutated targets.MethodsWe established five nasopharyngeal carcinoma (NPC) PDXs in mouse model. Subsequently, whole-exome sequencing (WES) and genomic mutation analyses were performed to search for genetic alterations for new drug targets. Potential drugs were applied in two NPC PDX mice model to assess their anti-cancer activities. RNA sequencing and transcriptomic analysis were performed in one NPC PDX mice to correlate with the efficacy of the anti-cancer drugs.ResultsA relative high incident rate of copy number variations (CNVs) of cell cycle-associated genes. Among the five NPC-PDXs, three had cyclin D1 (CCND1) amplification while four had cyclin-dependent kinase inhibitor CDKN2A deletion. Furthermore, CCND1 overexpression was observed in > 90% FFPE clinical metastatic NPC tumors (87/91) and was associated with poor outcomes. CNV analysis disclosed that plasma CCND1/CDKN2A ratio is correlated with EBV DNA load in NPC patients’ plasma and could serve as a screening test to select potential CDK4/6 inhibitor treatment candidates. Based on our NPC PDX model and RNA sequencing, Palbociclib, a cyclin-dependent kinase inhibitor, proved to have anti-tumor effects by inducing G1 arrest. One NPC patient with liver metastatic was treated with Palbociclib, had stable disease response and a drop in Epstein Barr virus (EBV) EBV titer.ConclusionsOur integrated information of sequencing-based genomic studies and tumor transcriptomes with drug treatment in NPC-PDX models provided guidelines for personalized precision treatments and revealed a cyclin-dependent kinase inhibitor Palbociclib as a novel candidate drug for NPC.

A Prognostic Bio-Model Based on SQSTM1 and N-Stage Identifies Nasopharyngeal Carcinoma Patients at High Risk of Metastasis for Additional Induction Chemotherapy.

Purpose: Metastasis is one of the most important causes of treatment failure in nasopharyngeal carcinoma (NPC). In T4 or N2-3 patients at high-risk of metastasis, concurrent chemoradiotherapy (CCRT) is inadequate and additional induction chemotherapy (IC) is controversial. There is a critical need to develop a better patient stratification to efficiently identify patients at high-risk of metastasis for additional IC. Recently, Sequestosome 1 (SQSTM1)/p62, an autophagy adaptor protein, was identified as one of the metastasis-related proteins in NPC. However, the mechanism by which SQSTM1 is involved in NPC metastasis was not investigated.Experimental Design: The effect of SQSTM1 on cell migration and invasion was examined in vitro and in vivo SQSTM1 expression was analyzed in clinical NPC samples using IHC. Luciferase reporter analyses were conducted to identify the effects of SQSTM1 on NF-κB transcriptional activity. A prediction bio-model was constructed by Cox analysis. Retrospective and prospective randomized clinical data were adopted to build and test the model, respectively.Results: SQSTM1 mediated epithelial to mesenchymal transition (EMT) through the NF-κB pathway to promote NPC metastasis. Inhibiting SQSTM1 enhanced sensitivity to cisplatin in NPC cells. In NPC patients, high SQSTM1 expression was associated with increased risk of distant metastasis. Furthermore, we propose a prognostic bio-model based on SQSTM1 and N-stage to predict NPC metastasis. Most importantly, our prospective randomized study suggested that IC is beneficial for NPC patients with high metastasis risk.Conclusions: The prognostic bio-model identifies NPC patients at high-risk of metastasis for additional IC. Clin Cancer Res; 24(3); 648-58. ©2017 AACR.

LncRNA HOTAIR contributes to the tumorigenesis of nasopharyngeal carcinoma via up-regulating FASN.

The therapeutic options for nasopharyngeal carcinoma (NPC) treatment have been restricted mainly to radiotherapy or chemotherapy, and the clinical treatment effect remains unsatisfactory. The primary purpose of this study was to investigate the molecular mechanisms of NPC and to find effective novel therapeutic targets for NPC. In order to analyze the expression level of lncRNA HOTAIR and FASN in human NPC clinical tissues or NPC cells, total RNA was extracted with TRIzol and the relative mRNA expression levels were detected by quantitative Real-time PCR. The endogenous expression of HOTAIR was modulated using lentivirus vectors transfection. The protein levels of Fatty acid synthase (FASN), p21 and MMP-9 in NPC cells were determined by Western blot when HOTAIR was knockdown. A free Fatty acid quantitation assay was performed to detect the intracellular free Fatty acid in NPC cells. The CCK-8 and colony formation assays were performed for cell viability and proliferation determination. The cell cycle of NPC cells was also determined by flow cytometric analysis. A matrigel invasion assay was performed to analyze the invasive ability of NPC cells. In this study, we observed a significant upregulation of lncRNA HOTAIR in NPC cells and clinical NPC specimens. The expression of Fatty acid synthase (FASN) was positively correlated to HOTAIR in NPC specimens. Knockdown of HOTAIR led to downregulation of FASN in NPC cells, thus suppressing cell proliferation and invasion. Additionally, de novo synthesis of cellular free fatty acid in NPC cells was inhibited when HOTAIR was knockdown. Furthermore, the protein levels of MMP-9 and p21 were downregulated when HOTAIR was knockdown. We suggest that HOTAIR is important in the progression and recurrence of NPC, perhaps through upregulating FASN. Targeting HOTAIR may be an effective therapeutic strategy for NPC.

The developmental transcription factor IRF6 attenuates ABCG2 gene expression and distinctively reverses stemness phenotype in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC), which originates from the nasopharynx, is highly prevalent in Southern China and Southeast Asia, and more than 90% of all NPCs are non-keratinizing undifferentiated cells or poorly differentiated squamous cells. Cancer stem cells (CSCs) are capable of self-renewal and have differentiation potential. These properties form the basis of cancer initiation, development, and radiochemoresistance. However, the molecular mechanisms underlying NPC CSC maintenance remain poorly understood. Here, genomic expression profiling using our previously established monoclonal cellular and animal models revealed that interferon regulatory factor 6 (IRF6) was downregulated in highly metastatic NPC cells, cancer stem-like NPC cells and animal models. Functional assays revealed that elevated IRF6 expression suppressed cell proliferation, growth, CSCs properties and enhanced cell chemotherapeutic sensitivity. However, silencing IRF6 resulted in opposing effects. Moreover, we determined that as a tumor suppressor gene and transcription factor, IRF6 directly bound the upstream region of the ATP-binding cassette sub-family G member 2 (ABCG2) DNA element and suppressed target ABCG2 expression in NPC cells. Consistently, an inverse correlation was observed between the mRNA levels of IRF6 and ABCG2 in clinical NPC samples. With these results, we provide the first evidence that IRF6 directly targets the ABCG2 gene and selectively kills CSCs in NPC and that IRF6 may be a valuable tool for developing new CSC-targeted treatment strategies for undifferentiated NPC patients.

MiR-125b Increases Nasopharyngeal Carcinoma Radioresistance by Targeting A20/NF-κB Signaling Pathway.

Radioresistance poses a major challenge in nasopharyngeal carcinoma (NPC) treatment, but little is known about how miRNA regulates this phenomenon. In this study, we investigated the function and mechanism of miR-125b in NPC radioresistance, one of upregulated miRNAs in the radioresistant NPC cells identified by our previous microarray analysis. We observed that miR-125b was frequently upregulated in the radioresistant NPCs, and its increment was significantly correlated with NPC radioresistance, and was an independent predictor for poor patient survival. In vitro radioresponse assays showed that miR-125b inhibitor decreased, whereas miR-125b mimic increased NPC cell radioresistance. In a mouse model, therapeutic administration of miR-125b antagomir dramatically sensitized NPC xenografts to irradiation. Mechanistically, we confirmed that A20 was a direct target of miR-125b and found that miR-125b regulated NPC cell radioresponse by targeting A20/NF-κB signaling. With a combination of loss-of-function and gain-of-function approaches, we further showed that A20 overexpression decreased while A20 knockdown increased NPC cell radioresistance both in vitro and in vivo Moreover, A20 was significantly downregulated while p-p65 (RelA) significantly upregulated in the radioresistant NPCs relative to radiosensitive NPCs, and miR-125b expression level was negatively associated with A20 expression level, whereas positively associated with p-p65 (RelA) level. Our data demonstrate that miR-125b and A20 are critical regulators of NPC radioresponse, and high miR-125b expression enhances NPC radioresistance through targeting A20 and then activating the NF-κB signaling pathway, highlighting the therapeutic potential of the miR-125b/A20/NF-κB axis in clinical NPC radiosensitization. Mol Cancer Ther; 16(10); 2094-106. ©2017 AACR.

Radiation-resistant cancer stem-like cell properties are regulated by PTEN through the activity of nuclear β-catenin in nasopharyngeal carcinoma.

Radiotherapy is the primary and most important treatment for nasopharyngeal carcinoma (NPC). Cancer stem-like cells (CSCs) have been shown to be resistant to radiation. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene has been suggested to play a role in stem cell self-renewal. In the present study, we sorted PTEN−/+ cells using a flow cytometer. The clone formation assay showed that PTEN− cells were more radioresistant than PTEN+ NPC cells. We found that PTEN− cells demonstrated a significant increase in tumorsphere formation and CSCs markers compared with PTEN+ cells. Silencing the expression of PTEN with siRNA resulted in increased expression of p-AKT, active β-catenin and Nanog. siPTEN cells irradiated showed more radioresistant and DNA damage than parental cells. We also confirmed that down-regulation of β-catenin expression with shRNA resulted in a reduced percentage of side population cells and expression of Nanog. shβ-catenin cells significantly decreased survivin expression at 4 Gy irradiation in PTEN− cells compared with PTEN+ cells. In siPTEN cells, β-catenin staining shifted from the cytoplasmic membrane to the nucleus. Furthermore, immunofluorescence showed that following irradiation of PTEN− cells, at 4 Gy, active β-catenin was mainly found in the nucleus. Immunohistochemistry analysis also demonstrated that the PTEN−/p-AKT+/β-catenin+/Nanog+ axis may indicate poor prognosis and radioresistance in clinical NPC specimens. Thus, our findings strongly suggest that PTEN− cells have CSCs properties that are resistant to radiation in NPC. PTEN exerts these effects through the downstream effector PI3K/AKT/β-catenin/Nanog axis which depends on nuclear β-catenin accumulation.

High FMNL3 expression promotes nasopharyngeal carcinoma cell metastasis: role in TGF-\u03b21-induced epithelia-to-mesenchymal transition

Formin-like 3 (FMNL3) plays a crucial role in cytoskeletal mediation and is potentially a biomarker for cell migration; however, its role in cancer metastasis remains unknown. In this study, we found elevated FMNL3 protein expression in clinical nasopharyngeal carcinoma (NPC) tissues. FMNL3 expression positively correlated to the clinical stage, T (tumour), N (lymph node metastasis) and M (distant metastasis) classification of NPC patients. Moreover, FMNL3 positively correlated to Vimentin expression and negatively correlated to E-cadherin expression in clinical NPC samples. In vitro experiments showed that FMNL3 expression was inversely related to NPC cell differentiation status. Overexpression of FMNL3 led to epithelial-to-mesenchymal transition (EMT) in well differentiated CNE1 cells. TGF-β1-treated poorly differentiated CNE2 cells showed changes in EMT accompanied by enhanced FMNL3 expression and cell migration. On the contrary, knockdown of FMNL3 partially attenuated the TGF-β1-promoted CNE2 cell migration, together with associated changes in EMT markers. Finally, knockdown of FMNL3 also weakened EMT in tumours in xenographs. Our study indicates for the first time that TGF-β1/FMNL3 signalling may be a novel mechanism mediating EMT in NPC, which is closely associated with NPC metastasis.

Functions of microRNA-143 in the apoptosis, invasion and migration of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a common cancer characterized by poor prognosis in areas of Southern China where it is endemic. microRNAs (miRNAs) are a class of naturally occurring small noncoding RNAs, some of which contribute to the initiation and development of cancer. The current study was designed to examine the expression level of miR-143 in NPC tissues. The potential functional targets of miR-143 involved in tumor apoptosis, invasion and migration were also investigated. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the expression levels of miR-143 in clinical NPC specimens. Western blotting was used to explore the expression levels of extracellular signal regulated kinase (ERK)-5, Kirsten rat sarcoma viral oncogene homolog (KRAS), caspase 3 and B-cell lymphoma 2 (Bcl-2) in CNE-2Z cells following transfection with miR-143. Significantly decreased expression levels of miR-143 were observed in NPC tissues in comparison with matched normal nasopharyngeal tissues. Moreover, negative associations of miR-143 with tumor invasion depth, as well as lymph node metastasis were found. The enforced expression of miR-143 induced NPC cell apoptosis in addition to the suppression of growth, migration and invasion. The functions of miR-143 in NPC are mediated, at least in part, by the inhibition of ERK-5 activity and promotion of caspase 3 and KRAS expression. These findings suggest that miR-143 may function as a tumor suppressor in the development and progression of NPC.

T4/N2 classification nasopharyngeal carcinoma benefit from concurrent chemotherapy in the era of intensity-modulated radiotherapy.

Although the benefits of concurrent chemotherapy (CC) in the treatment of locally advanced nasopharyngeal carcinoma (NPC) had been proven in the era of two-dimensional radiotherapy, long-term efficacy and safety of using CC combined with intensity-modulated radiotherapy (IMRT) remain unclear. A retrospective analysis of 1,182 patients who underwent IMRT for clinical II-Iva NPC was performed. Propensity score matching algorithm was used to identify two matched cohorts with or without CC (264 patients per cohort). Median follow-up time was 45.6 and 43.6 months for the two cohorts. The estimated 5-year overall survival rate was 81.8% (95% CI 76.6-87.4) in patients treated with CC and 73.7% (95% CI 67.8-80.0) in those treated without CC, respectively (hazard ratio 0.64, 95% CI 0.44–0.93; p = 0.018). The benefit of CC was mainly observed in those patients with good performance status, male, age > 48 years, T4 and N2 classification. Grade 3/4 acute toxicities were more common in those patients administrated with CC. The grade and incidence of late salivary glands damage were also increased by CC (p = 0.003). These findings indicated that the addition of CC significantly improved treatment outcomes of NPC patients treated with IMRT, but accompanied increased toxicities. Tailored CC and optimizing schedule of IMRT and systemic therapy were needed, provided that distant metastasis was the predominant pattern of failure in patients treated with IMRT.

Importance of β-Catenin for cancer stem cell generation and tumorigenic activity in nasopharyngeal carcinoma.

e17532Background: Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors with poor prognosis and recurrence in South China. The hard eradication of NPC in clinic is predominantly...

The putative tumor activator ARHGEF3 promotes nasopharyngeal carcinoma cell pathogenesis by inhibiting cellular apoptosis.

Nasopharyngeal carcinoma (NPC) is one of the most prevalent forms of highly invasive malignancy in Southern China and Southeast Asia. The pathogenesis of NPC is a multistep process driven by the acquisition of numerous genetic abnormalities. We investigated the potential oncogenic role of the Rho-guanine nucleotide exchange factor 3 gene, ARHGEF3, in NPC pathogenesis. Expression levels of ARHGEF3 were frequently up-regulated in NPC cell lines and tissues. In a large cohort of clinical NPC tissues high expression of ARHGEF3 was positively associated with an increased T status, distant metastasis, and a more advanced clinical stage (P < 0.05). Survival analysis revealed that ARHGEF3 expression was a significant and independent prognosis factor for NPC patients. In NPC cell lines, knockdown of ARHGEF3 was sufficient to inhibit cell growth, motility, and invasion in vitro, whereas ectopic overexpression of ARHGEF3 substantially enhanced NPC cells tumorigenesis and metastasis in vivo. Depletion of ARHGEF3 in NPC cells dramatically promoted caspase-3 induced apoptosis and an anti-apoptosis factor, BIRC8, was identified as a critical downstream target of the ARHGEF3. Our findings suggest that increased expression of ARHGEF3 plays a critical oncogenic role in NPC pathogenesis by preventing cell apoptosis through the up-regulation of BIRC8, and ARHGEF3 might be employed as a novel prognostic marker and effective therapeutic target for human NPC.

β-Catenin is important for cancer stem cell generation and tumorigenic activity in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors with poor prognosis and recurrence in South China. The hard eradication of NPC in clinic is predominantly due to cancer stem cells (CSCs). Increasing evidence revealed that the aberrant activation of Wnt/β-catenin was positively correlated with the produce of CSCs. To further investigate the effect of β-catenin on CSCs and tumorigenesis in NPC, a CNE2 cell line (pLKO.1-sh-β-catenin-CNE2) with stably suppressed expression of β-catenin was used in this study. The expressions of biomarkers in CSCs including c-myc, Nanog, Oct3/4, Sox2, EpCAM as well as adhesion-related proteins like E-cadherin and vimentin were analyzed by western blot analysis and immunofluorescent staining. The proliferation and migration abilities were investigated by MTT assay and Transwell assay, respectively. Cell cycle was analyzed by flow cytometry. Finally, xenograft was performed to determine the effect of β-catenin on oncogenesis in vivo. Results showed that the expressions of c-myc, Nanog, Oct3/4, Sox2, and EpCAM were all decreased in pLKO.1-sh-β-catenin-CNE2 cells. It was also found that vimentin was downregulated, while E-cadherin was upregulated. Results of MTT and Transwell assays suggested that the proliferation and migration abilities were impaired by silencing of β-catenin, and more cells were arrested in G1 phase when compared with the control. In vivo study indicated that the tumor growth was markedly suppressed in experimental group. Based on current findings, β-catenin may function as an essential protein for the maintenance of migration and proliferation abilities of NPC cells, and a complicated network consisting of c-myc, Nanog, Oct3/4, Sox2, EpCAM, E-cadherin, vimentin, and β-catenin may be involved in the inherent regulation mechanisms.

A new staging system for nasopharyngeal carcinoma based on intensity-modulated radiation therapy: results of a prospective multicentric clinical study

PurposeTo establish a new clinical staging standard for nasopharyngeal carcinoma (NPC), based on intensity-modulated radiotherapy (IMRT), through a prospective multicenter clinical trial.Experiment Design492 NPC patients were selected from six hospitals in the Guangxi Zhuang Autonomous Region, China from January 2006 to December 2009. Kaplan-Meier method was adopted to calculate survival rates. Log-rank test was used to compare survival differences.ResultsAccording to the seventh edition of the UICC/AJCC staging system, the differences between T1, T2 and T3 are not statistically significant, suggesting that T1, T2 and T3 could be combined as new T1. There were significant differences between all N stages except those of N3a and N3b, suggesting that N3a and N3b could be combined as new N3. Additionally, the overall survival (OS) curves of stages I, II, III and IVa were not significantly different. Therefore, we propose a new clinical NPC staging standard based on magnetic resonance imaging (MRI) and IMRT as T stage (including T1 and T2), N stage (including N0, N1, N2 and N3) and clinical staging includes I (T1N0M0), II (T1N1-2M0, T2N0M0), III (T2N1-2M0), IVa (TxN3M0) and IVb (TxNxM1). Recommended staging system performs better in risk difference and distribution balance. Furthermore, the differences in the 5-year curves of local relapse-free survival (LRFS), distant metastasis-free survival (DMFS), and OS were all statistically more significant than the seventh edition of the UICC/AJCC staging system.ConclusionsProposed staging system is more adaptable to IMRT and predicts the prognosis of NPC patients more accurately.

Long noncoding RNA Hotair mediated angiogenesis in nasopharyngeal carcinoma by direct and indirect signaling pathways.

Nasopharyngeal carcinoma (NPC), as a unique head and neck cancer type, is particularly prevalent in certain geographic areas such as eastern Asia. Until now, the therapeutic options have been restricted mainly to radiotherapy or chemotherapy. However, the clinical treatment effect remains unsatisfactory even if the combined radio-chemotherapies. Therefore, it is urgently needed to develop effective novel therapies against NPC. In this study, we discovered that lncRNA Hotair was extremely abundant in NPC cells and clinical NPC samples. Further studies showed that Hotair knockdown significantly attenuated both in vitro and in vivo tumor cell growth and angiogenesis. Our study also demonstrated that Hotair promoted angiogenesis through directly activating the transcription of angiogenic factor VEGFA as well as through GRP78-mediated upregulation of VEGFA and Ang2 expression. Therefore, Hotair may serve as a promising diagnostic marker and therapeutic target for NPC patients.