Methylation-associated silencing of miR-9-1 promotes nasopharyngeal carcinoma progression and glycolysis via HK2.

Characteristically, cancer cells metabolize glucose through aerobic glycolysis, known as the Warburg effect. Accumulating evidence suggest that during cancer formation, microRNAs (miRNAs) could regulate such metabolic reprogramming. In the present study, miR-9-1 was identified as significantly hypermethylated in nasopharyngeal carcinoma (NPC) cell lines and clinical tissues. Ectopic expression of miR-9-1 inhibited NPC cell growth and glycolytic metabolism, including reduced glycolysis, by reducing lactate production, glucose uptake, cellular glucose-6-phosphate levels, and ATP generation in vitro and tumor proliferation in vivo. HK2 (encoding hexokinase 2) was identified as a direct target of miR-9-1 using luciferase reporter assays and Western blotting. In NPC cells, hypermethylation regulates miR-9-1 expression and inhibits HK2 translation by directly targeting its 3' untranslated region. MiR-9-1 overexpression markedly reduced HK2 protein levels. Restoration of HK2 expression attenuated the inhibitory effect of miR-9-1 on NPC cell proliferation and glycolysis. Fluorescence in situ hybridization results indicated that miR-9-1 expression was an independent prognostic factor in NPC. Our findings revealed the role of the miR-9-1/HK2 axis in the metabolic reprogramming of NPC, providing a potential therapeutic strategy for NPC.

Effects of Txk­mediated activation of NF­κB signaling pathway on neurological deficit and oxidative stress after ischemia­reperfusion in rats.

Ischemic stroke is an extremely mortal cerebrovascular disease, and neuroinflammation and oxidative stress emerge as important traits of ischemic stroke. However, as an inflammation­associated factor, Txk tyrosine kinases (Txk) has been poorly studied in neuroscience research. The aim of the present study was to investigate the role of Txk after ischemia­reperfusion (I/R) in vivo and in vitro, observe the association between Txk knockdown and neurological deficit and oxidative stress, and to explore whether the process was mediated by the activation of nuclear factor (NF)­κB signaling pathway. Middle cerebral artery occlusion (MCAO), oxygen and glucose deprivation/reperfusion (OGD/R) model and western blotting have been used to simulate the I/R injury to analyze the expression, and to approximate the localization of Txk, respectively. Brain infarct volume, neurological score, brain water content, apoptosis and oxidative stress assays in vivo and apoptosis, cellular viability, the LDH release and oxidative stress assays in vitro were observed using a Txk­knockdown lentivirus. Finally, NF­κB overexpression lentivirus was applied to discuss whether the role of Txk following I/R was regulated by the NF­κB signaling pathway. The results show that the Txk expression peaked at 24 h after MCAO and 6 h after OGD/R, respectively. Txk molecules gradually entered the nucleus after MCAO and OGD/R. The Txk­knockdown lentivirus resulted in decreased brain infarct volume, neurological score, brain water content, apoptosis and oxidative stress after MCAO in vivo. Besides, Txk knockdown decreased apoptosis, LDH release, oxidative stress, and increased cellular viability, after ODG in vitro. Finally, NF­κB overexpression reversed the process of neurological deficit and oxidative stress after Txk regulation in vivo and vitro. Overall, the present study suggests that Txk potentially regulates apoptosis, neurological deficit, and oxidative stress after I/R, by entering the nucleus. NF­κB maybe the downstream target factor of Txk.

Gemcitabine plus cisplatin versus fluorouracil plus cisplatin as a first-line concurrent chemotherapy regimen in nasopharyngeal carcinoma: a prospective, multi-institution, randomized controlled phase II study.

BACKGROUND: The objective of this study was to evaluate the efficacy and safety of gemcitabine plus cisplatin concurrent chemoradiotherapy (CCRT) in patients with nasopharyngeal carcinoma. METHOD: Patients with NPC were randomly assigned to the gemcitabine plus cisplatin (GP) group or fluorouracil plus cisplatin (PF) group. Primary end-point was disease-free survival (DFS); secondary endpoints: overall survival, distant metastasis-free survival (DMFS), locoregional relapse-free survival, and treatment-related adverse events. RESULTS: Seventy-six patients were prospectively enrolled and the median follow-up time was 41 months (9-61 months). Three-year DFS were similar between the GP and PF groups (73.7% vs. 60.5%, HR 0.66, 95% CI 0.30-1.44; P = 0.30). Distant metastasis was the most common failure form in PF compared with GP (P = 0.034). Three-year DMFS was significantly better in the GP group than PF group (89.5% vs. 71.1%, P = 0.045). Grade 3-4 gastrointestinal toxicities (vomiting and diarrhea) were significantly more common in the PF group; grade 3-4 neutropenia and thrombocytopenia were more common in the GP group. CONCLUSION: Gemcitabine plus cisplatin could be used as an alternative regimen in CCRT for nasopharyngeal carcinoma.

Candidate tumor suppressor ZNF154 suppresses invasion and metastasis in NPC by inhibiting the EMT via Wnt/ß-catenin signalling.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is especially prevalent in southeast Asia and southern China, but its molecular mechanisms remain poorly characterized. DNA methylation is associated with initiation and progression of tumors, including NPC. Through a genome-wide DNA methylation screening approach, we discovered ZNF154, but its methylation status and roles in NPC have not been investigated. METHODS: The methylation status of ZNF154 in NPC was detected with Methylation specific-PCR (MSP) and Quantitative Sequenom MassARRAY. The invasion and migration capacities were examined by wound healing and transwell invasion assays. The role of ZNF154 in NPC metastasis was clarified with experimental metastasis assay in vivo. Western blotting analysis was used to investigate protein changes followed by ZNF154 over-expression. Kaplan-Meier analysis was performed to determine the association between ZNF154 methylation and prognosis in NPC. RESULTS: Compared to immortalized nasopharyngeal tissues and cells, ZNF154 expression was frequently downregulated in NPC tissues and cell lines due to promoter methylation. Demethylation treatment with 5-aza-2-deoxycytidine (5-Aza) restored ZNF154 expression in NPC cell lines. Ectopic overexpression of ZNF154 in NPC cells inhibited cell migration and invasion in vitro and lung nodule formation in an in vivo tumor metastasis assay. Mechanistic investigations suggested ZNF154 inhibits Wnt/ß-catenin signalling pathway activation and prevents the EMT in NPC. Furthermore, Kaplan-Meier analysis showed hypermethylation of the ZNF154 promoter was associated with significantly poorer disease-free survival (P = 0.032) and distant metastasis-free survival (P = 0.040) among patients with locoregionally advanced NPC. CONCLUSIONS: Taken together, these findings define a novel role for ZNF154 as a tumor suppressor in NPC.

Matched analysis of induction chemotherapy plus chemoradiotherapy versus induction chemotherapy plus radiotherapy alone in locoregionally advanced nasopharyngeal carcinoma: a multicenter study.

BACKGROUND: The relative efficacy of induction chemotherapy (IC) followed by concurrent chemoradiotherapy (CCRT) versus IC followed by radiotherapy (RT) alone in locoregionally advanced NPC remains unclear. METHODS: A total of 877 patients with locally advanced NPC who underwent IC/CCRT or IC/RT at four institutions in China between January 2004 and December 2010 were retrospectively assessed. IC was cisplatin-based combination chemotherapy; concurrent chemotherapy, single agent cisplatin. Two-dimensional conventional radiotherapy (2DCRT) was the radiotherapy technique. All patients were matched in an equal ratio using a pair-matched method. Overall survival (OS), disease-free survival (DFS), distant metastasis-free survival (DMFS), locoregional relapse-free survival (LRRFS) and toxicities were assessed. RESULTS: Eligible patients were matched (n = 642; 321 patients per arm) based on eight clinicopathological characteristics. Five-year OS, DFS, DMFS, and LRRFS were 76%, 70%, 86%, and 88% for IC/CCRT and 75%, 70%, 90%, and 91% for IC/RT, respectively. There were no statistically significant survival differences between arms (P>0.05), even in subgroup analysis. In multivariate analysis, treatment (IC/CCRT vs. IC/RT) was not an independent prognostic factor for any survival end-point. Grade 3/4 acute gastrointestinal toxicities (vomiting, nausea) and hematological toxicities (leucopenia/neutropenia, thrombocytopenia and anemia) were significantly more common in the IC/CCRT arm than IC/RT arm during RT. CONCLUSION: Overall, IC/CCRT failed to demonstrate any survival advantage but higher acute toxicities over IC/RT in locoregionally advanced NPC.