DCAF7 Acts as A Scaffold to Recruit USP10 for G3BP1 Deubiquitylation and Facilitates Chemoresistance and Metastasis in Nasopharyngeal Carcinoma.

Despite docetaxel combined with cisplatin and 5-fluorouracil (TPF) being the established treatment for advanced nasopharyngeal carcinoma (NPC), there are patients who do not respond positively to this form of therapy. However, the mechanisms underlying this lack of benefit remain unclear. DCAF7 is identified as a chemoresistance gene attenuating the response to TPF therapy in NPC patients. DCAF7 promotes the cisplatin resistance and metastasis of NPC cells in vitro and in vivo. Mechanistically, DCAF7 serves as a scaffold protein that facilitates the interaction between USP10 and G3BP1, leading to the elimination of K48-linked ubiquitin moieties from Lys76 of G3BP1. This process helps prevent the degradation of G3BP1 via the ubiquitin‒proteasome pathway and promotes the formation of stress granule (SG)-like structures. Moreover, knockdown of G3BP1 successfully reversed the formation of SG-like structures and the oncogenic effects of DCAF7. Significantly, NPC patients with increased levels of DCAF7 showed a high risk of metastasis, and elevated DCAF7 levels are linked to an unfavorable prognosis. The study reveals DCAF7 as a crucial gene for cisplatin resistance and offers further understanding of how chemoresistance develops in NPC. The DCAF7-USP10-G3BP1 axis contains potential targets and biomarkers for NPC treatment.

LncRNA DYNLRB2-AS1 promotes gemcitabine resistance of nasopharyngeal carcinoma by inhibiting the ubiquitination degradation of DHX9 protein.

Gemcitabine (GEM) based induction chemotherapy is a standard treatment for locoregionally advanced nasopharyngeal carcinoma (NPC). However, approximately 15 % of patients are still resistant to GEM-containing chemotherapy, which leads to treatment failure. Nevertheless, the underlying mechanisms of GEM resistance remain poorly understood. Herein, based on a microarray analysis, we identified 221 dysregulated lncRNAs, of which, DYNLRB2-AS1 was one of the most upregulated lncRNAs in GEM-resistance NPC cell lines. DYNLRB2-AS1 was shown to function as contain an oncogenic lncRNA that promoted NPC GEM resistance, cell proliferation, but inhibited cell apoptosis. Mechanistically, DYNLRB2-AS1 could directly bind to the DHX9 protein and prevent its interaction with the E3 ubiquitin ligase PRPF19, and thus blocking PRPF19-mediated DHX9 degradation, which ultimately facilitated the repair of DNA damage in the presence of GEM. Clinically, higher DYNLRB2-AS1 expression indicated an unfavourable overall survival of NPC patients who received induction chemotherapy. Overall, this study identified the oncogenic lncRNA DYNLRB2-AS1 as an independent prognostic biomarker for patients with locally advanced NPC and as a potential therapeutic target for overcoming GEM chemoresistance in NPC.

PJA1-mediated suppression of pyroptosis as a driver of docetaxel resistance in nasopharyngeal carcinoma.

Chemoresistance is a main reason for treatment failure in patients with nasopharyngeal carcinoma, but the exact regulatory mechanism underlying chemoresistance in nasopharyngeal carcinoma remains to be elucidated. Here, we identify PJA1 as a key E3 ubiquitin ligase involved in nasopharyngeal carcinoma chemoresistance that is highly expressed in nasopharyngeal carcinoma patients with nonresponse to docetaxel-cisplatin-5-fluorouracil induction chemotherapy. We find that PJA1 facilitates docetaxel resistance by inhibiting GSDME-mediated pyroptosis in nasopharyngeal carcinoma cells. Mechanistically, PJA1 promotes the degradation of the mitochondrial protein PGAM5 by increasing its K48-linked ubiquitination at K88, which further facilitates DRP1 phosphorylation at S637 and reduced mitochondrial reactive oxygen species production, resulting in suppression of GSDME-mediated pyroptosis and the antitumour immune response. PGAM5 knockdown fully restores the docetaxel sensitization effect of PJA1 knockdown. Moreover, pharmacological targeting of PJA1 with the small molecule inhibitor RTA402 enhances the docetaxel sensitivity of nasopharyngeal carcinoma in vitro and in vivo. Clinically, high PJA1 expression indicates inferior survival and poor clinical efficacy of TPF IC in nasopharyngeal carcinoma patients. Our study emphasizes the essential role of E3 ligases in regulating chemoresistance and provides therapeutic strategies for nasopharyngeal carcinoma based on targeting the ubiquitin-proteasome system.

Transcription factor ATMIN facilitates chemoresistance in nasopharyngeal carcinoma.

Despite that the docectaxel-cisplatin-5-fluorouracil (TPF) induction chemotherapy has greatly improved patients' survival and became the first-line treatment for advanced nasopharyngeal carcinoma (NPC), not all patients could benefit from this therapy. The mechanism underlying the TPF chemoresistance remains unclear. Here, by analyzing gene-expression microarray data and survival of patients who received TPF chemotherapy, we identify transcription factor ATMIN as a chemoresistance gene in response to TPF chemotherapy in NPC. Mass spectrometry and Co-IP assays reveal that USP10 deubiquitinates and stabilizes ATMIN protein, resulting the high-ATMIN expression in NPC. Knockdown of ATMIN suppresses the cell proliferation and facilitates the docetaxel-sensitivity of NPC cells both in vitro and in vivo, while overexpression of ATMIN exerts the opposite effect. Mechanistically, ChIP-seq combined with RNA-seq analysis suggests that ATMIN is associated with the cell death signaling and identifies ten candidate target genes of ATMIN. We further confirm that ATMIN transcriptionally activates the downstream target gene LCK and stabilizes it to facilitate cell proliferation and docetaxel resistance. Taken together, our findings broaden the insight into the molecular mechanism of chemoresistance in NPC, and the USP10-ATMIN-LCK axis provides potential therapeutic targets for the management of NPC.

The E3 ligase NEURL3 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma by promoting vimentin degradation.

BACKGROUND: Metastasis has emerged as the major reason of treatment failure and mortality in patients with nasopharyngeal carcinoma (NPC). Growing evidence links abnormal DNA methylation to the initiation and progression of NPC. However, the precise regulatory mechanism behind these processes remains poorly understood. METHODS: Bisulfite pyrosequencing, RT-qPCR, western blot, and immunohistochemistry were used to test the methylation and expression level of NEURL3 and its clinical significance. The biological function of NEURL3 was examined both in vitro and in vivo. Mass spectrometry, co-immunohistochemistry, immunofluorescence staining, and ubiquitin assays were performed to explore the regulatory mechanism of NEURL3. RESULTS: The promoter region of NEURL3, encoding an E3 ubiquitin ligase, was obviously hypermethylated, leading to its downregulated expression in NPC. Clinically, NPC patients with a low NEURL3 expression indicated an unfavorable prognosis and were prone to develop distant metastasis. Overexpression of NEURL3 could suppress the epithelial mesenchymal transition and metastasis of NPC cells in vitro and in vivo. Mechanistically, NEURL3 promoted Vimentin degradation by increasing its K48-linked polyubiquitination at lysine 97. Specifically, the restoration of Vimentin expression could fully reverse the tumor suppressive effect of NEURL3 overexpression in NPC cells. CONCLUSIONS: Collectively, our study uncovers a novel mechanism by which NEURL3 inhibits NPC metastasis, thereby providing a promising therapeutic target for NPC treatment.

DNAJA4 suppresses epithelial-mesenchymal transition and metastasis in nasopharyngeal carcinoma via PSMD2-mediated MYH9 degradation.

Emerging evidence indicates that DNA methylation plays an important role in the initiation and progression of nasopharyngeal carcinoma (NPC). DNAJA4 is hypermethylated in NPC, while its role in regulating NPC progression remains unclear. Here, we revealed that the promoter of DNAJA4 was hypermethylated and its expression was downregulated in NPC tissues and cells. Overexpression of DNAJA4 significantly suppressed NPC cell migration, invasion, and EMT in vitro, and markedly inhibited the inguinal lymph node metastasis and lung metastatic colonization in vivo, while it did not affect NPC cell viability and proliferation capability. Mechanistically, DNAJA4 facilitated MYH9 protein degradation via the ubiquitin-proteasome pathway by recruiting PSMD2. Furthermore, the suppressive effects of DNAJA4 on NPC cell migration, invasion, and EMT were reversed by overexpression of MYH9 in NPC cells. Clinically, a low level of DNAJA4 indicated poor prognosis and an increased probability of distant metastasis in NPC patients. Collectively, DNAJA4 serves as a crucial driver for NPC invasion and metastasis, and the DNAJA4-PSMD2-MYH9 axis might contain potential targets for NPC treatments.

TRIM21 inhibits irradiation-induced mitochondrial DNA release and impairs antitumour immunity in nasopharyngeal carcinoma tumour models.

Although radiotherapy can promote antitumour immunity, the mechanisms underlying this phenomenon remain unclear. Here, we demonstrate that the expression of the E3 ubiquitin ligase, tumour cell-intrinsic tripartite motif-containing 21 (TRIM21) in tumours, is inversely associated with the response to radiation and CD8+ T cell-mediated antitumour immunity in nasopharyngeal carcinoma (NPC). Knockout of TRIM21 modulates the cGAS/STING cytosolic DNA sensing pathway, potentiates the antigen-presenting capacity of NPC cells, and activates cytotoxic T cell-mediated antitumour immunity in response to radiation. Mechanistically, TRIM21 promotes the degradation of the mitochondrial voltage-dependent anion-selective channel protein 2 (VDAC2) via K48-linked ubiquitination, which inhibits pore formation by VDAC2 oligomers for mitochondrial DNA (mtDNA) release, thereby inhibiting type-I interferon responses following radiation exposure. In patients with NPC, high TRIM21 expression was associated with poor prognosis and early tumour relapse after radiotherapy. Our findings reveal a critical role of TRIM21 in radiation-induced antitumour immunity, providing potential targets for improving the efficacy of radiotherapy in patients with NPC.

LINC00173 facilitates tumor progression by stimulating RAB1B-mediated PA2G4 and SDF4 secretion in nasopharyngeal carcinoma.

An increasing number of studies have found that long non-coding RNA (lncRNA) play important roles in driving the progression of nasopharyngeal carcinoma (NPC). Our microarray screening revealed that expression of the lncRNA long intergenic non-protein coding RNA 173 (LINC00173) was upregulated in NPC. However, its role and mechanism in NPC have not yet been elucidated. In this study, we demonstrate that high LINC00173 expression indicated a poor prognosis in NPC patients. Knockdown of LINC00173 significantly inhibited NPC cell proliferation, migration and invasion in vitro. Mechanistically, LINC00173 interacted and colocalized with Ras-related protein Rab-1B (RAB1B) in the cytoplasm, but the modulation of LINC00173 expression did not affect the expression of RAB1B at either the mRNA or protein levels. Instead, relying on the stimulation of RAB1B, LINC00173 could facilitate the extracellular secretion of proliferation-associated 2G4 (PA2G4) and stromal cell-derived factor 4 (SDF4; also known as 45-kDa calcium-binding protein) proteins, and knockdown of these proteins could reverse the NPC aggressive phenotype induced by LINC00173 overexpression. Moreover, in vivo LINC00173-knockdown models exhibited a marked slowdown in tumor growth and a significant reduction in lymph node and lung metastases. In summary, LINC00173 serves as a crucial driver for NPC progression, and the LINC00173-RAB1B-PA2G4/SDF4 axis might provide a potential therapeutic target for NPC patients.

Association of Intratumoral Microbiota With Prognosis in Patients With Nasopharyngeal Carcinoma From 2 Hospitals in China.

Importance: Microbiota-tumor interactions have qualified microbiota as a promising prognostic biomarker in various types of cancers. Although the nasopharynx acts as a crucial niche of the upper respiratory tract microbiome, whether the intratumoral microbiota exists and its clinical significance in nasopharyngeal carcinoma (NPC) remain uncertain. Objective: To evaluate the clinical significance of intratumoral microbiota for individual prognostication in patients with NPC. Design, Setting, and Participants: This retrospective cohort study included NPC biopsy samples from 2 hospitals: Sun Yat-sen University Cancer Center (Guangzhou, China) and Zhejiang Cancer Hospital (Hangzhou, China) between January 2004 and November 2016, with follow-up through November 2020. A total of 802 patients were included according to the following criteria: with histologically proven NPC, without distant metastasis at initial diagnosis, had not received antitumor treatment before biopsy sampling, aged between 18 and 70 years, with complete medical records and regular follow-up, without a history of cancer, and successfully extracted enough DNA for experiments. Main Outcomes and Measures: The primary end point was disease-free survival, and the secondary end points included distant metastasis-free survival and overall survival. To assess the existence and load of intratumoral microbiota in 96 patients with NPC with or without tumor relapse, 16S rRNA sequencing and quantitative polymerase chain reaction were used. The associations between intratumoral bacterial load and clinical outcome were evaluated in 241 fresh-frozen NPC samples (training cohort) and validated in paraffin-embedded NPC samples of internal (n = 233) and external (n = 232) validation cohorts. Metagenomic and transcriptome analyses were performed to ascertain the origin and underlying mechanism of intratumoral bacteria. Results: A total of 802 patients with NPC (mean [SD] age, 46.2 [10.6] years; 594 [74.1%] male) were enrolled. Microbiota presented within NPC tumor tissues, among which Corynebacterium and Staphylococcus predominated. Patients with a high bacterial load in the training cohort had inferior rates of disease-free survival (hazard ratio [HR], 2.90; 95% CI, 1.72-4.90; P < .001), distant metastasis-free survival (HR, 3.18; 95% CI, 1.58-6.39; P < .001), and overall survival (HR, 3.41; 95% CI, 1.90-6.11, P < .001) than those with a low bacterial load, a finding that was validated by the internal and external validation cohorts. Single-nucleotide variant analysis revealed that the nasopharyngeal microbiota was the main origin of NPC intratumoral bacteria. Transcriptome and digital pathology analyses demonstrated that a higher intratumoral bacterial load was negatively associated with T-lymphocyte infiltration. Conclusions and Relevance: Intratumoral bacterial load was a robust prognostic tool for patients with NPC in this cohort study, indicating potential guidance for treatment decisions in patients at different levels of risk of malignant progression.

A lncRNA signature associated with tumor immune heterogeneity predicts distant metastasis in locoregionally advanced nasopharyngeal carcinoma.

Increasing evidence has revealed the roles of long noncoding RNAs (lncRNAs) as tumor biomarkers. Here, we introduce an immune-associated nine-lncRNA signature for predicting distant metastasis in locoregionally advanced nasopharyngeal carcinoma (LA-NPC). The nine lncRNAs are identified through microarray profiling, followed by RT-qPCR validation and selection using a machine learning method in the training cohort (n = 177). This nine-lncRNA signature classifies patients into high and low risk groups, which have significantly different distant metastasis-free survival. Validations in the Guangzhou internal (n = 177) and Guilin external (n = 150) cohorts yield similar results, confirming that the signature is an independent risk factor for distant metastasis and outperforms anatomy-based metrics in identifying patients with high metastatic risk. Integrative analyses show that this nine-lncRNA signature correlates with immune activity and lymphocyte infiltration, which is validated by digital pathology. Our results suggest that the immune-associated nine-lncRNA signature can serve as a promising biomarker for metastasis prediction in LA-NPC.

Gemcitabine synergizes with cisplatin to inhibit nasopharyngeal carcinoma cell proliferation and tumor growth.

In a recently published phase III clinical trial, gemcitabine (GEM) plus cisplatin (DDP) induction chemotherapy significantly improved recurrence-free survival and overall survival and became the standard of care among patients with locoregionally advanced NPC. However, the molecular mechanisms of GEM synergized with DPP in NPC cells remain elucidated. These findings prompt us to explore the effect of the combination between GEM and DDP in NPC cell lines through proliferative phenotype, immunofluorescence, flow cytometry, and western blotting assays. In vitro studies reveal that GEM or DPP treated alone induces cell cycle arrest, promotes cell apoptosis, forces DNA damage response, and GEM synergism with DDP significantly increases the above effects in NPC cells. In vivo studies indicate that GEM or DPP treated alone significantly inhibits the tumor growth and prolongs the survival time of mice injected with SUNE1 cells compared to the control group. Moreover, the mice treated with GEM combined with DDP have smaller tumors and survive longer than those in GEM or DPP treated alone group. In addition, P-gp may be the key molecule that regulates the synergistic effect of gemcitabine and cisplatin. GEM synergizes with DPP to inhibit NPC cell proliferation and tumor growth by inducing cell cycle arrest, cell apoptosis, and DNA damage response, which reveals the mechanisms of combined GEM and DDP induction chemotherapy in improving locoregionally advanced NPC.

Prognostic Value of Pretreatment Serum Cystatin C Level in Nasopharyngeal Carcinoma Patients in the Intensity-modulated Radiotherapy Era.

PURPOSE: Serum cystatin C has been considered as a significant prognostic factor for various malignancies. This study aimed to evaluate the relationship between serum cystatin C level before antitumor treatment and the prognosis of nasopharyngeal carcinoma (NPC) patients treated with intensity-modulated radiotherapy (IMRT). PATIENTS AND METHODS: A cohort of 2077 NPC patients were enrolled between April 2009 and September 2012. The Kaplan-Meier curves and log rank tests were used to determine the differences of overall survival (OS) and disease-free survival (DFS). Univariate and multivariate Cox regression analyses were used to determine independent prognostic factors. RESULTS: Overall, 362/2077 (17.4%) patients had high serum cystatin C level, and they were older and more male (both P<0.001), and they had higher TNM stage (all P<0.05). Kaplan-Meier analysis revealed that patients with high serum cystatin C had worse OS (P<0.001) and DFS (P<0.001). Univariate and multivariate Cox regression analysis showed that high serum cystatin C level was an independent prognostic predictor of OS (HR: 1.56, 95%CI: 1.25-1.95) and DFS (HR: 1.38, 95%CI: 1.13-1.68). Subgroup analysis based on TNM stage revealed that advanced-stage NPC patients with high serum cystatin C had poorer OS (P<0.001) and DFS (P<0.001). CONCLUSION: Our results revealed that high serum cystatin C level before antitumor treatment can predict clinical outcomes of NPC patients treated with IMRT, and it can guide clinicians to formulate more personalized therapy for NPC patients.

Serum Calcium Levels Before Antitumour Therapy Predict Clinical Outcomes in Patients with Nasopharyngeal Carcinoma.

PURPOSE: The prognostic value of serum calcium levels in nasopharyngeal carcinoma (NPC) remains unknown. This study aimed to evaluate the prognostic value of serum calcium levels in patients with NPC. PATIENTS AND METHODS: A total of 2094 patients diagnosed with NPC between April 2009 and September 2012 were enrolled in this retrospective analysis. The median follow-up time was 96.3 months (range: 4.1-120.0 months). Univariate and multivariable Cox proportional hazards models were used to identify significant and independent prognostic predictors of overall survival (OS), disease-free survival (DFS), distant metastasis-free survival (DMFS), and relapse-free survival (RFS). RESULTS: Overall, low serum calcium levels were detected in 1109/2094 (53.00%) patients and tended to be more frequently detected in older (P<0.001) and female (P=0.001) patients. Patients with low serum calcium levels had poorer OS (P=0.011), DFS (P=0.012) and DMFS (P=0.004) than those with high serum calcium levels, but serum calcium levels had no significant effect on RFS (P=0.376). In univariate and multivariable analyses, low serum calcium levels were a statistically significant and independent prognostic factor for OS, DFS, and DMFS but had no prognostic value for RFS. CONCLUSION: Serum calcium levels can serve as a prognostic predictor and guide more individualized treatment for NPC patients.

AR-induced long non-coding RNA LINC01503 facilitates proliferation and metastasis via the SFPQ-FOSL1 axis in nasopharyngeal carcinoma.

Increasing evidence indicates that long non-coding RNAs (lncRNAs) play vital roles in the tumorigenesis and progression of cancers. However, the functions and regulatory mechanisms of lncRNAs in nasopharyngeal carcinoma (NPC) are still largely unknown. Our previous lncRNA expression profiles identified that LINC01503 was overexpressed in NPC. Here, we verified that LINC01503 was highly expressed in NPC and correlated with poor prognosis. LINC01503 promoted NPC cell proliferation, migration, and invasion in vitro, and facilitated tumor growth and metastasis in vivo. Mechanistically, LINC01503 recruited splicing factor proline-and glutamine-rich (SFPQ) to activate Fos like 1 (FOSL1) transcription, and ectopic expression of FOSL1 reversed the suppressive effect of LINC01503 knockdown on NPC progression. Moreover, androgen receptor (AR)-mediated transcription activation was responsible for the overexpression of LINC01503, and AR ligand-dependent cell growth, migration, and invasion in NPC cells. Taken together, our findings reveal that AR-induced LINC01503 can promote NPC progression through the SFPQ-FOSL1 axis, which represents a novel prognostic biomarker and therapeutic target for NPC patients.

FNDC3B 3'-UTR shortening escapes from microRNA-mediated gene repression and promotes nasopharyngeal carcinoma progression.

Alternative polyadenylation (APA), which induces shortening of the 3'-UTR, is emerging as an important feature in cancer development and progression. Nevertheless, the effects and mechanisms of APA-induced 3'-UTR shortening in nasopharyngeal carcinoma (NPC) remain largely unclear. Fibronectin type III domain containing 3B (FNDC3B) tended to use proximal polyadenylation site and produce shorter 3'-UTR according to our previous sequencing study. Herein, we found that FNDC3B with shorter 3'-UTR could escape from miRNA-mediated gene repression, and caused its increased expression in NPC. Knocking down of FNDC3B inhibited NPC cell proliferation, migration, invasion, and metastasis in vitro and in vivo. Overexpression of FNDC3B, especially those with shorter 3'-UTR, promoted NPC progression. Furthermore, the mechanism study revealed that FNDC3B could bind to and stabilize myosin heavy chain 9 (MYH9) to activate the Wnt/ß-catenin signaling pathway. In addition, MYH9 could reverse the inhibitory effects of FNDC3B knockdown in NPC. Altogether, our results suggested that the 3'-UTR shortening of FNDC3B mRNA mediated its overexpression in NPC and promoted NPC progression by targeting MYH9. This newly identified FNDC3B-MYH9-Wnt/ß-catenin axis could represent potential targets for individualized treatment in NPC.