Electrophysiological Changes on Laryngeal Motor Neuropathways Cause Voice Disorders for Postradiotherapy Patients with Nasopharyngeal Carcinoma.

OBJECTIVE: This study explored electrophysiological changes in the laryngeal motor neuropathway and determined whether lesions in the laryngeal motor cortex (LMC) and its descending tract contribute to voice deterioration and peripheral nerve palsy in patients with nasopharyngeal carcinoma (NPC) postradiotherapy (RT). STUDY DESIGNS: Prospective cohort study. METHODS: Twenty-two patients with NPC at 2 to 4years post-RT (8 female and 14 male), 22 patients with NPC at 8 to 10years post-RT (8 female and 14 male), and 22 healthy individuals (9 female and 13 male) were selected to test their magnetic evoked potentials (MEP), motor nerve conduction, and voice quality using transcranial magnetic stimulation, laryngeal electromyography, and the XION DiVAS acoustic analysis software. Three groups were matched according to approximate age. Multiple comparisons were performed among the three groups. RESULTS: The voice quality of post-RT patients with NPC deteriorated compared to that of healthy individuals. Bilateral LMC and their corticonuclear tracts to the bilateral ambiguous nuclei of post-RT patients with NPC were impaired according to multigroup comparisons of MEP amplitudes, latencies, and resting motor thresholds. The vagus and recurrent laryngeal nerves (RLN) of post-RT patients with NPC were impaired according to multigroup comparisons of the amplitude and latencies of the compound muscle action potential and latencies of f-waves. CONCLUSIONS: The voice quality of patients with NPC deteriorated after RT. The pathogenesis of post-RT voice deterioration may involve radiation-induced injuries to the vagus, RLN, and bilateral LMC. Furthermore, radiation-induced injuries to the bilateral LMC may contribute to vagus and RLN palsies. These findings support the use of transcranial approaches to treating voice disorders and peripheral nerve palsies in post-RT patients with NPC. TRIAL REGISTRATION: ChiCTR2100054425; Electrophysiological Study of Vocal-Fold Mobility Disorders After Radiotherapy for NPC Patients via Magnetic Evoked Potential and Their Correlation with Voice Quality Assessment; https://www.chictr.org.cn/bin/project/edit?pid=144429.

NPM1 promotes nasopharyngeal carcinoma metastasis and stemness by recruiting Mdm2 to induce the ubiquitination-mediated degradation of p53.

Nasopharyngeal carcinoma (NPC) is clinically challenging due to the development of distant metastasis following initial therapy. Therefore, it is necessary to elucidate the mechanisms underlying metastases to develop novel therapeutic strategies. Nucleophosmin 1 (NPM1) has been directly linked to the development of human tumors and may have both tumor-suppressing and oncogenic properties. Although NPM1 is often overexpressed in solid tumors of various histopathological origins, its specific function in mediating the development of NPC is still unknown. Here, we investigated the role of NPM1 in NPC and discovered that NPM1 was elevated in clinical NPC samples and served as a predictor of the worst prognosis in NPC patients. Furthermore, the upregulation of NPM1 promoted the migration and the cancer stemness of NPC both in vitro and in vivo. Mechanistic analyses revealed that the E3 ubiquitin ligase Mdm2 was recruited by NPM1 to induce the ubiquitination-mediated proteasomal degradation of p53. Ultimately, knockdown of NPM1 suppressed the stemness and EMT signals. In summation, this study demonstrated the role and the underlying molecular mechanism of NPM1 in NPC, providing the evidence for the clinical application of NPM1 as a therapeutic target for the treatment of patients with NPC.

Deriving prognostic significance from a molecular subtype model of laryngeal carcinoma.

BACKGROUND: This study explored whether laryngeal carcinoma could be divided into different subtypes based on molecular differences using a molecular subtype-prediction model. METHODS: We extracted data from the Cancer Genome Atlas and Gene Expression Omnibus databases and then performed unsupervised cluster analysis to identify discrete molecular subtypes of laryngeal carcinoma. Significance analysis of microarrays was performed to detect differentially expressed genes for each subtype, and gene set enrichment analysis and the GenCliP3 software were used to label gene functions and identify key pathways. RESULTS: We categorized 126 patients into C1 and C2 molecular subtypes associated with pathologic grade. The C2 subtype appeared more aggressive, with a worse prognosis. The most significant enrichment pathway of the C2 subtype was the Hedgehog pathway, and GLI1 was a core gene. CONCLUSIONS: Laryngeal carcinoma can be divided into two subtypes based on differences in molecular expression, which could identify key molecules associated with prognosis.

Insufficient PINX1 expression stimulates telomerase activation by direct inhibition of EBV LMP1-NF-κB axis during nasopharyngeal carcinoma development.

OBJECTIVE: Early malignant transformation of nasopharyngeal carcinoma(NPC) is associated with Epstein-Barr virus(EBV) infection and telomerase activation. The EBV latent membrane protein 1(LMP1) regulates expression of various genes by triggering NF-κB signaling pathway. PINX1 is a well-identified tumor suppressor gene by inhibiting telomerase activity and cancer cell growth. However, whether and how EBV inhibit PINX1 expression and activate telomerase in NPC is still incompletely elucidated. METHODS: Immunohistochemistry, real-time PCR and Western blotting were utilized to explore the expression of PINX1. Chromatin immunoprecipitation(ChIP) and Dual-luciferase reporter assay were used to elucidate the regulatory mechanism between NF-κB and PINX1. TRAP-SYBR Green assay and Southern blotting were utilized to detect telomerase activity and telomere length. CCK8 and EdU tests were conducted to measure proliferation ability. RESULTS: We demonstrated that PINX1 is down-regulated in NPC for the first time. Mechanistically, we found that LMP1 could inhibit the transcriptional activity of PINX1 by promoting the binding of p65 to three specific sites in PINX1 promoter, significantly, two(-1698/-1689, tgcaatttcc; -206/-197, cgggctttac) of which have not been reported. In addition, we also observed that LMP1 overexpression resulted in increased telomerase activity, prolonged telomere length and enhanced proliferation. CONCLUSION: We first discovered EBV led to reduced PINX1 expression through LMP1-NF-κB-PINX1 axis, which up-regulated telomerase activity in NPC. And hence, the tumor cells acquired the ability to proliferate more exuberantly. This signaling pathway illustrates the relationship between EBV latent infection and telomerase activation, and further provides new thinking for early diagnosis and treatment in NPC.

Maximal number of pre-synaptic ribbons are formed in cochlear region corresponding to middle frequency in mice.

OBJECTIVE: To investigate whether there are more quantitative pre-synaptic ribbons formed in the cochlear region corresponding to middle-frequency in cochlea of mice. METHODS: Counts of pre-synaptic ribbons were performed using immunostaining and laser confocal microscopy. Hearing thresholds and function of ribbon synapses were estimated by auditory brain response (ABR) and compound action potential (CAP). Cochlear mapping has been achieved to match the frequencies and corresponding regions along the cochlear spiral. RESULTS: The number of pre-synaptic ribbons in per inner hair cell (IHC) has been found to increase gradually from the base turn, the maximal quantity appeared at the region of 50-70% from the apex. Next, ABR thresholds showed that there was the lowest ABR threshold in the frequency around 8-16 kHz, corresponding to the region of 50-70% from the apex according to the cochlear mapping. Further, CAP amplitudes were estimated, and the maximal value identified at the same frequency (8-16 kHz). CONCLUSIONS: Maximal number of pre-synaptic ribbons is formed in the cochlear region of middle frequency in mice, coupling with the lowest ABR threshold and highest CAP amplitudes. Our study shows that the middle frequency (8-16 kHz) could be the most sensitive region to sound stimuli in mice.

Higher methylation intensity induced by EBV LMP1 via NF-κB/DNMT3b signaling contributes to silencing of PTEN gene.

Phosphatase and tensin homolog (PTEN) is a major tumor suppressor and usually silenced via the deletion, insertion and mutation. We previously discovered its inactivation via aberrant CpG island methylation. Here, we provide further evidence that EBV latent membrane protein 1(LMP1) can induce a higher intensity of DNA methylation at PTEN CpG islands, inactivating PTEN at the cellular and molecular level. Initially, increased methylation intensity of PTEN CpG islands was observed in EBV-infected nasopharyngeal carcinoma (NPC) cells, accompanied by decreased PTEN expression. In NPC tissue samples showing the methylation at PTEN promoter, LMP1 was highly expressed in higher methylation intensity group relative to lower intensity group, and DNA methyltransferase 3b (DNMT3b) expression was positively correlated with LMP1 expression. Moreover, transfection of LMP1 gene into EBV-negative NPC cells demonstrated that LMP1 up-regulated DNMT3b expression, leading to a higher intensity of PTEN CpG island methylation. Mechanistically, computational prediction and luciferase reporter assay identified a functional NF-κB binding site on DNMT3b promoter and the mutated NF-κB binding site abolished LMP1-mediated DNMT3b activation. Chromatin immunoprecipitation displayed that NF-κB p65 subunit constitutively bound to DNMT3b promoter, supporting the activation of DNMT3b by EBV LMP1 via NF-κB signaling. Furthermore, the expression level of DNMT3b was observed to be increased in the nuclei of LMP1-expressing NPC cells, and a NF-κB inhibitor, PDTC, counteracted LMP1-mediated DNMT3b overexpression. Thus, this study first reports that LMP1-mediated NF-κB can up-regulate DNMT3b transcription, thereby leading to relatively higher methylation intensity at PTEN CpG islands, and ultimately silencing major tumor suppressor PTEN.

The important role of the receptor for activated C kinase 1 (RACK1) in nasopharyngeal carcinoma progression.

BACKGROUND: The receptor for activated C kinase 1 (RACK1) is involved in various cancers, but its roles in nasopharyngeal carcinoma (NPC) have not yet been fully elucidated. METHODS: Initially, RACK1 expression was analyzed by immunohistochemistry in NPC and normal nasopharyngeal (NP) tissues. It was also detected by qPCR and Western blot in NPC cells. Confocal microscope and immunofluorescence were performed to detect the subcellular compartmentalization of RACK1. Subsequently, after up- or down-regulating RACK1 in NPC cells, cell proliferation and migration/invasion were tested using in vitro assays including MTT, EdU, colony formation, Transwell and Boyden assays. Furthermore, several key molecules were detected by Western blot to explore underlying mechanism. Finally, clinical samples were analyzed to confirm the relationship between RACK1 expression and clinical features. RESULTS: Receptor for activated C kinase 1 expression was much higher in NPC than NP tissues. And RACK1 was mainly located in the cytoplasm. Overexpression of RACK1 promoted NPC cell proliferation and metastasis/invasion, whereas depletion of this protein suppressed NPC cell proliferation and metastasis/invasion. Mechanistically, RACK1 deprivation obviously suppressed the activation of Akt and FAK, suggesting the PI3K/Akt/FAK pathway as one of functional mechanisms of RACK1 in NPC. Furthermore, clinical sample analysis indicated a positive correlation between in vivo expression of RACK1 with lymph node invasion and clinical stage of NPC. CONCLUSION: Our results demonstrate that RACK1 protein plays an important role in NPC development and progression. The upregulation of RACK1 can promote the proliferation and invasion of NPC by regulating the PI3K/Akt/FAK signal pathway. Thus, this study contributes to the discovery of a potential therapeutic target for NPC.

Aberrant CpG island methylation of PTEN is an early event in nasopharyngeal carcinoma and a potential diagnostic biomarker.

The inactivation of phosphatase and tensin homolog (PTEN) due to its CpG island hypermethylation has been observed in some types of tumors except nasopharyngeal carcinoma (NPC). In the present study, we focused on the aberrant methylation of PTEN CpG islands in NPC. The mRNA expression of PTEN was detected by quantitative PCR in 45 NPC and 22 non-tumor nasopharyngeal epithelial (NP) tissues. The methylation status of PTEN was examined by methylation-specific polymerase chain reaction and sequencing. The mRNA expression of PTEN in three NPC cell lines treated with 5-aza-2'-deoxycytidine (5-aza-dC) was also examined. PTEN was downregulated in both NPC tissues and NPC cell lines and a relatively higher methylation level of PTEN was found in NPC specimens (82.2%) relative to NP tissues (5.3%). The PTEN mRNA expression was restored in NPC cell lines by treatment with 5-aza-dC. These results first reveal an epigenetic alteration, aberrant methylation of PTEN, in NPC, which is probably an early event and may be regarded as a novel candidate biomarker for early stage of NPC detection and prevention.