Radioresistance Of Nasopharyngeal Carcinoma Cells Research Articles

TRAF4 regulates ubiquitination-modulated survivin turnover and confers radioresistance.

Nasopharyngeal carcinoma (NPC) is the most common cancer originating in the nasopharynx. Despite continuous improvement in treatment strategies, recurrence or persistence of cancer after radiotherapy is still inevitable, highlighting the need to identify therapeutic resistance factors and develop effective methods for NPC treatment. Herein, we found that TRAF4 is overexpressed in NPC cells and tissues. Knockdown TRAF4 significantly increased the radiosensitivity of NPC cells, possibly by inhibiting the Akt/Wee1/CDK1 axis, thereby suppressing survivin phosphorylation and promoting its degradation by FBXL7. TRAF4 is positively correlated with p-Akt and survivin in NPC tissues. High protein levels of TRAF4 were observed in acquired radioresistant NPC cells, and knockdown of TRAF4 overcomes radioresistant in vitro and the xenograft mouse model. Altogether, our study highlights the TRAF4-survivin axis as a potential therapeutic target for radiosensitization in NPC.

Elevation of H3K27me3 level contributes to the radioresistance of nasopharyngeal carcinoma by inhibiting OAS1 expression.

Radiotherapy has long been a main treatment option for nasopharyngeal carcinoma (NPC). However, during clinical treatment, NPC is prone to developing radioresistance, resulting in treatment failure. This study aims to examine the role of histone methylation in the induction of radioresistance. It was found that the radioresistance of NPC cells was related to the increase of the level of histone H3 lysine 27 trimethylation (H3K27me3). Treatment of cells with histone methyltransferase inhibitor GSK126 increased the radiosensitivity of NPC cells by triggering Bcl2 apoptosis regulator/BCL2-associated X, apoptosis regulator (Bcl2/BAX) signaling pathway. Bioinformatics analysis indicated that the expression of 2'-5'-oligoadenylate synthetase 1 (OAS1) was reduced in the radioresistant cells but increased in the GSK126-treated cells. Chromatin immunoprecipitation assay confirmed that the decrease of OAS1 expression in radioresistant cells was mainly due to the enrichment of H3K27me3 in its promoter region. Furthermore, downregulation of OAS1 reduced apoptosis due to the inhibition of Bcl2/BAX pathway after irradiation, while OAS1 overexpression increased radiosensitivity. Our findings revealed for the first time that the increase of H3K27me3 level was associated with the decrease of OAS1 expression, leading to the inhibition of apoptosis and ultimately contributing to the radioresistance of NPC cells. Moreover, the histone methyltransferase inhibitor GSK126 could overcome the radioresistance and thus might be a potential therapeutic strategy for NPC.NEW & NOTEWORTHY Our findings revealed for the first time that the increase of H3K27me3 level was associated with the decrease of OAS1 expression, leading to the inhibition of apoptosis and ultimately contributing to the radioresistance of NPC cells. Moreover, we demonstrated that the histone methyltransferase inhibitor GSK126 could be a promising therapeutic strategy for NPC by overcoming radioresistance, providing valuable insights into the clinical treatment of NPC.

Inhibition of MNK pathway sensitizes nasopharyngeal carcinoma to radiotherapy.

Improving the clinical management of nasopharyngeal carcinoma (NPC) is an unmet need owing to the high incidence of treatment failure caused by radioresistance. In our study, we observed increased phosphorylation of translation initiation factor 4E (eIF4E), regulated by MAP kinase-interacting kinase (MNK), in NPC cells following irradiation treatment. Using siRNA to deplete MNK, we found that radiation-induced eIF4E phosphorylation was eliminated, NPC cell sensitivity to radiation was enhanced, and radioresistant NPC cell viability was reduced. Furthermore, we tested three pharmacological MNK inhibitors (eFT508, CGP57380, and cercosporamide) and found that they were effective against radioresistant NPC cells and synergized with irradiation. In-vivo experiments confirmed that eFT508, at a tolerable dose, inhibited the growth of radioresistant NPC and synergized with radiation in a radiosensitive NPC xenograft model. Our research highlights the activation of MNK-mediated survival mechanisms in NPC in response to radiotherapy and the potential of combining radiation with MNK inhibitors as a sensitizing strategy. Notably, eFT508 is currently being investigated in clinical trials for cancer treatment, and our findings may prompt the initiation of clinical trials using eFT508 in radioresistant NPC patients.

Long Non-Coding RNA LINC00202 Enhances Radiotherapy Sensitivity of Nasopharyngeal Carcinoma by Regulating Ras and Rab Interactor 1

Radiotherapy resistance is a major problem faced in clinical treatment of nasopharyngeal carcinoma (NPC). This study aims to illustrate the role of LINC00202 in influencing radiotherapy resistance of NPC patients. Microarray analyses on differentially expressed long non-coding RNAs (lncRNAs) and their target miRNAs and mRNAs in the profiling containing radiotherapy resistant and sensitive NPC tissues were conducted. The biological functions of LINC00202 in regulating proliferative ability, cell cycle progression and apoptosis in irradiated HNE-2 cells were explored. In addition, in vivo effects of LINC00202 on NPC growth were examined by establishing the xenograft models. LINC00202 was downregulated in radiotherapy resistant NPC tissues and irradiation-induced radio resistant NPC cells. In vitro experiments showed that overexpression of LINC00202 in HNE-2 cells suppressed viability and induced cell cycle arrest in G1 phase. Survival rate of HNE-2 cells overexpressing LINC00202 dose-dependently decreased following different doses of irradiation. Consistently, 4-Gy irradiation in mice with in vivo expression of LINC00202 presented slower tumor growth of NPC than those of controls. Microarray analyses uncovered the LINC00202/miR-513A-5P/RIN1 axis, which was responsible for enhancing radiotherapy sensitivity of NPC. LINC00202/miR-513a-5p/RIN1 axis is responsible for enhancing radiotherapy sensitivity of NPC, which provides a new idea in clinical treatment of NPC.

Long noncoding RNA LINC00173 induces radioresistance in nasopharyngeal carcinoma via inhibiting CHK2/P53 pathway.

Radiotherapy is the backbone of nasopharyngeal carcinoma (NPC), nearly 11-17% NPC patients suffered local relapse and 18-37% suffered distant metastasis mainly due to radioresistance. Therefore, the key of improving patients' survivals is to investigate the mechanism of radioresistance. In this study, we revealed that the expression level of long intergenic nonprotein coding RNA 173 (LINC00173) was significantly increased in the radioresistant NPC patients' tumour tissues compared with the radiosensitive patients by RNA-sequencing, which also predict poor prognosis in NPC. Overexpression of LINC00173 induced radioresistance of NPC cells in vitro and in vivo. Mechanistically, LINC00173 bound with checkpoint kinase 2 (CHK2) in nucleus, and impaired the irradiation-induced CHK2 phosphorylation, then suppressed the activation of P53 signalling pathway, which eventually inhibiting apoptosis and leading to radioresistance in NPC cells. In summary, LINC00173 decreases the occurrence of apoptosis through inhibiting the CHK2/P53 pathway, leads to NPC radioresistance and could be considered as a novel predictor and therapeutic target in NPC.

YAP nuclear translocation facilitates radiation resistance in nasopharyngeal carcinoma cells

ObjectivesInvestigate the role of the Hippo-YAP signaling pathway in radioresistant Nasopharyngeal Carcinoma (NPC). MethodsEstablishment of radioresistant CNE-1 cells (CNE-1-RR) by gradually increasing ionizing radiation (IR) doses, and identifying the apoptosis of CNE-1-RR by flow cytometry. We employed immunoblot and immunofluorescence staining to detect the expression of YAP in both CNE-1-RR and control group cells. Moreover, we validated the role of YAP in CNE-1-RR by inhibiting its nuclear translocation. ResultsIn contrast to the control group, radioresistant NPC cells demonstrated significant YAP dephosphorylation and nuclear translocation. CNE-1-RR cells exhibited enhanced activation of γ-H2AX (Ser139) upon exposure to IR and greater recruitment of double-strand breaks (DSBs) repair-related proteins. Additionally, inhibiting YAP nuclear translocation in radioresistant CNE-1-RR cells significantly increased their sensitivity to radiotherapy. ConclusionsThe present investigation has unveiled the intricate mechanisms and physiological roles of YAP in CNE-1-RR cells exhibiting resistance to IR. Based on our findings, it can be inferred that a combinational therapeutic strategy involving radiotherapy and inhibitors that impede the nuclear translocation of YAP holds promising potential for treating radioresistant NPC.

Knockdown of LAMB3 suppressed radioresistance in nasopharyngeal carcinoma via deactivating NRF2 signaling pathway.

Nasopharyngeal carcinoma (NPC) is a prevalent malignancy in Southeast Asia and Southern China. Laminin subunit beta-3 (LAMB3) has been validated to participate in diverse cancers. Nevertheless, the role and mechanism of LAMB3 in NPC remain unclear. In this study, LAMB3 expression is upregulated in NPC cells and tissues. Interestingly, knockdown of LAMB3 promoted apoptosis and reduced the radioresistance of NPC cells. Besides, shLAMB3 enhanced X-ray-induced reactive oxygen species (ROS) accumulation. Mechanically, knockdown of LAMB3 deactivated nuclear factor erythroid-2-related factor 2 (NRF2) signaling pathway via enhancing forkhead box 3 (FOXO3) expression. In rescue experiments, suppression of NRF2 signaling pathway abrogated shLAMB3-induced NPC cell apoptosis and ROS accumulation under X-ray treatment. Similarly, LAMB3 knockdown restrains NPC tumor growth and reduces radioresistance in vivo. Thus, these findings concluded that knockdown of LAMB3 enhanced apoptosis and ROS accumulation, and suppressed radioresistance in NPC via enhancing FOXO3 expression and deactivating NRF2 signaling pathway, facilitating the development of novel strategies for NPC radioresistance.

Genome-scale CRISPR-Cas9 knockout screening in nasopharyngeal carcinoma for radiosensitive and radioresistant genes

BackgroundGenome-scale CRISPR-Cas9 knockout screening may provide new insights into the mechanism underlying clinical radioresistance in nasopharyngeal carcinoma (NPC), which is remain largely unknown. Our objective was to screen the functional genes associated with radiosensitivity and radioresistance in NPC, laying a foundation for further research on its functional mechanismand. MethodsCRISPR-Cas9 library lentivirus screening in radiation-treated NPC cells was combined with second-generation sequence technology to identify functional genes, which were further validated in radioresistant NPC cells and patient tissues. ResultsEleven radiosensitive and radioresistant genes were screened. Among these genes, the expression of FBLN5, FAM3C, MUS81, and DNAJC17 were significantly lower and TOMM20, CDKN2AIP, SNX22, and SP1 were higher in the radioresistant NPC cells (C666-1R, 5-8FR) (p < 0.05). CALD1 was highly expressed in C666-1R. Furthermore, we found knockout of FBLN5, FAM3C, MUS81 and DNAJC17 promoted the proliferation of NPC cells, while CDKN2AIP and SP1 had the opposed results (p < 0.05). This result was verified in NPC patient tissues. Meanwhile, KEGG analysis showed that the Fanconi anemia pathway and the TGF-β signaling pathway possibly contributed to radiosensitivity or radioresistance in NPC. ConclusionsNine genes involved in the radiosensitivity or radioresistance of NPC: four genes for radiosensitivity (FBLN5, FAM3C, MUS81, and DNAJC17), two genes for radioresistance (CDKN2AIP, SP1), two potential radioresistant genes (TOMM20, SNX22), and a potential radiosensitive gene (CALD1). Genome-scale CRISPR-Cas9 knockout screening for radiosensitive and radioresistant genes in NPC may provide new insights into the mechanisms underlying clinical radioresistance to improve the efficacy of radiotherapy for NPC.

LncRNA CASC19 Enhances the Radioresistance of Nasopharyngeal Carcinoma by Regulating the miR-340-3p/FKBP5 Axis.

Radioresistance remains a serious obstacle encountered in the radiotherapy of nasopharyngeal carcinoma (NPC). Both mRNAs and non-coding RNAs (ncRNAs), including long ncRNA (lncRNA) and microRNA (miRNA), play essential roles in radiosensitivity. However, the comprehensive expression profiles and competing endogenous RNA (ceRNA) regulatory networks among lncRNAs, miRNAs, and mRNAs in NPC radioresistance are still bewildering. In this study, we performed an RNA-sequencing (RNA-seq) assay in the radioresistant NPC cells CNE2R and its parental cells CNE2 to identify the differentially expressed lncRNAs, miRNAs, and mRNAs. The ceRNA networks containing lncRNAs, miRNAs, and mRNAs were predicted on the basis of the Pearson correlation coefficients and authoritative miRanda databases. In accordance with bioinformatic analysis of the data of the tandem mass tag (TMT) assay of CNE2R and CNE2 cells and the gene chip assay of radioresistant NPC samples in pre- and post-radiotherapy, the radioresistance-related signaling network of lncRNA CASC19, miR-340-3p, and FKBP5 was screened and further verified using an RT-qPCR assay. CASC19 was positively associated with FKBP5 expression while negatively correlated with miR-340-3p, and the target binding sites of CASC19/miR-340-3p and miR-340-3p/FKBP5 were confirmed using a dual-luciferase reporter assay. Moreover, using an mRFP-GFP-LC3 maker, it was found that autophagy contributed to the radioresistance of NPC. MiR-340-3p inhibition or FKBP5 overexpression could rescue the suppression of autophagy and radioresistance induced by CASC19 knockdown in CNE2R cells. In conclusion, the CASC19/miR-340-3p/FKBP5 network may be instrumental in regulating NPC radioresistance by enhancing autophagy, which provides potential new therapeutic targets for NPC.

Knockdown of lncRNA SNHG16 Attenuates the Proliferation and Radioresistance of Nasopharyngeal Carcinoma Cells by Mediating miR-31-5p/SFN Axis.

Nasopharyngeal carcinoma (NPC) is a rare head and neck tumor that threatens people's health. Radiotherapy is a major treatment for NPC, however, radioresistance of the NPC cells may contribute to treatment failure. LncRNA SNHG16 was upregulated in NPC; however, the function of SNHG16 in radioresistant NPC cells remains unexplored. RT-qPCR was applied for detecting SNHG16, miR-31-5p and SFN levels. MTT assay and colony formation assay were applied to assess the cell viability and proliferation. Dual luciferase was applied for assessing the relation among SNHG16, miR-31-5p and SFN. SFN level in NPC cells was examined by Western blot. The level of SNHG16 and SFN in NPC cells was significantly upregulated by exposure to radiation. In addition, silencing of SNHG16 or miR-31-5p mimics notably attenuated radioresistance of NPC cells. SNHG16 could positively regulate the expression of SFN in NPC cells through binding with miR-31-5p. Furthermore, SNHG16 downregulation obviously attenuated the proliferation and radioresistance of NPC cells by regulation of miR-31-5p/SFN axis. Knockdown of lncRNA SNHG16 attenuates radioresistance of nasopharyngeal carcinoma cells by miR-31-5p/SFN axis. Thus, our research data show a novel method for improving the efficacy of radiotherapy for NPC.

Extracellular matrix stiffness mediates radiosensitivity in a 3D nasopharyngeal carcinoma model

PurposeRadiotherapy is one of the essential treatment modalities for nasopharyngeal carcinoma (NPC), however, radioresistance still poses challenges. Three-dimensional (3D) tumor culture models mimic the in vivo growth conditions of cells more accurately than 2D models. This study is to compare the tumor biological behaviors of NPC cells in 2D, On-Surface 3D and Embedded 3D systems, and to investigate the correlation between radioresistance and extracellular matrix (ECM) stiffness.MethodsThe morphology and radioresistance of the human NPC cell line CNE-1 were observed in 2D and 3D systems. The CCK-8 assay, wounding healing assays, flow cytometry, soft agar assays, and western blot analysis were used to evaluate differences in biological behaviors such as proliferation, migration, cell cycle distribution, and stem cell activity. Different ECM stiffness systems were established by co-blending collagen and alginate in varying proportions. ECM stiffness was evaluated by compressive elastic moduli measurement and colony formation assay was used to assess radioresistance of NPC cells in systems with different ECM stiffness after irradiation.ResultsCompared to 2D models, the morphology of NPC cells in 3D culture microenvironments has more in common with in vivo tumor cells and 3D cultured NPC cells exhibit stronger radioresistance. Integrin β1 but not the epithelial-to-mesenchymal transition pathway in 3D models boost migration ability. Cell proliferation was enhanced, the proportion of tumor stem cells was increased, and G1/S phase arrest occurred in 3D models. NPC cells cultured in softer ECM systems (with low alginate proportions) exhibit striking resistance to ionizing radiation.ConclusionThe tumor biological behaviors of NPC cells in 3D groups were obviously different from that of 2D. Radioresistance of NPC cells increased with the stiffness of ECM decreasing.

M6A demethylase FTO renders radioresistance of nasopharyngeal carcinoma via promoting OTUB1-mediated anti-ferroptosis

Radiotherapy is a valid treatment for nasopharyngeal carcinoma (NPC), and radioresistance is the main cause of local NPC treatment failure. However, the underlying mechanisms and valuable markers of radioresistance for NPC remain have not been established. In this study, we observed that the m6A mRNA demethylase fat mass and obesity-associated protein (FTO) was significantly upregulated in radioresistant NPC tissues and cells relative to parental radiosensitive NPC tissues and cells. FTO enhances radioresistance by repressing radiation-induced ferroptosis in NPC. Mechanistically, FTO acts as an m6A demethylase to erase the m6A modification of the OTUB1 transcript and promote the expression of OTUB1, thereby inhibiting the ferroptosis of cells induced by radiation and finally triggering the radiotherapy resistance of NPC. Furthermore, our in vivo experiment results showed that the FTO inhibitor, FB23-2, and the ferroptosis activator, erastin, altered tumor responsiveness to radiotherapy in NPC cell lines and patient-derived xenografts. Our findings reveal, for the first time, that FTO enhances NPC radiotherapy resistance by withstanding radiation-induced ferroptosis, suggesting that FTO may serve as a potential therapeutic target and valuable prognostic biomarker in patients with NPC.

Downregulation of annexin A3 promotes ionizing radiation-induced EGFR activation and nuclear translocation and confers radioresistance in nasopharyngeal carcinoma

Radioresistance currently poses a significant challenge to successful disease control of nasopharyngeal carcinoma (NPC). We previously uncovered that annexin A3 (ANXA3), a calcium-dependent phospholipid binding protein, is underexpressed in radioresistant NPC cells and mouse xenografts. This study aims to further unravel the mechanistic basis underlying ANXA3-mediated radioresistance in NPC. We show that either innate ANXA3 downregulation or short hairpin RNA(shRNA)-based knockdown of ANXA3 confers resistance to ionizing radiation (IR) in NPC both in vitro and in mouse xenograft models in vivo, whereas radiosensitization was observed when ANXA3 was ectopically expressed. Mechanistically, ANXA3 knockdown dramatically enhances IR-induced epidermal growth factor receptor (EGFR) phosphorylation and nuclear translocation, leading to increased post-IR phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) concomitant with markedly accelerated DNA DSB repair. In addition, pretreatment with cetuximab efficiently abrogated the radioresistant phenotype of ANXA3-low cells as well as the ANXA3 knockdown-induced post-IR EGFR nuclear accumulation, suggesting that EGFR is an essential mediator for ANXA3 depletion-mediated radioprotection in NPC. Collectively, this work reveals for the first time a critical role of ANXA3 in radiation survival and DNA repair mechanism of NPC and provides mechanistic evidence to support ANXA3 as a potential therapeutic target to improve radiocurability for NPC.

C2orf40 inhibits metastasis and regulates chemo-resistance and radio-resistance of nasopharyngeal carcinoma cells by influencing cell cycle and activating the PI3K/AKT/mTOR signaling pathway.

BackgroundNasopharyngeal carcinoma (NPC) is a malignant tumor of epithelial origin in head and neck with high incidence rate in Southern China. C2orf40 has been identified as a tumor suppressor gene in many cancers. However, the roles of C2orf40 in nasopharyngeal carcinoma has not been studied.MethodsIn this study, a bioinformatics analysis was performed to identify the differentially expressed genes in NPC. The quantitative methylation levels was detected using pyrosequencing. qRT-PCR, western blotting, immunohistochemistry and immunofluorescence were used to detect the expression level of related RNA and proteins. Cell proliferation was detected using CCK-8 assay, and colony formation capability was detected using colony formation assays. Cell migration and invasion were analyzed using wound-healing and Transwell assays, respectively. The apoptosis level of cells was assessed using TUNEL staining. Endogenous DNA damage and repair were assessed by the comet assay. Cell cycle analyses carried out by flow cytometry. Finally, We used a xenograft nude mouse to verify the roles of C2orf40 in chemoresistance and radioresistance in vivo.ResultsWe found that the C2orf40 expression was significantly downregulated in NPC tissues and inversely associated with a poor prognosis. In vivo and in vitro functional experiments confirmed that overexpression of C2orf40 significantly inhibited the migration and invasion of NPC cells, and promoted their sensitivity to radiotherapy and chemotherapy of NPC cells. Mechanically, the expression level of C2orf40 was negatively correlated with the expression levels of CCNE1 and CDK1. Overexpression of C2orf40 induced cell cycle arrest of NPC cells at G/M phase. In addition, C2orf40 can down-regulated the expression levels of homologous recombination-related proteins (BRCA1, BRCA2, RAD51, and CDC25A) and inhibited the activity of the PI3K/AKT/mTOR signaling pathway.ConclusionThe results clarified the biological functions and mechanisms of C2orf40, as a tumor suppressor gene, in NPC, and provided a potential molecular target for improving the sensitivity of NPC to radiotherapy and chemotherapy.

Inhibition of PDK1 enhances radiosensitivity and reverses epithelial-mesenchymal transition in nasopharyngeal carcinoma.

Radioresistance challenges the clinical outcomes of nasopharyngeal carcinoma (NPC). The 3-phosphoinositide-dependent protein kinase 1 (PDK1) is a crucial kinase of PI3K/AKT signaling pathway which has been implicated in the process of radioresistance. However, the role of PDK1 in NPC remains largely unclear. The expression of PDK1 was determined by immunohistochemistry and Western blot. The effects of RNA interference and pharmacologic inhibitor of PDK1 in combination with irradiation were investigated. Overexpression of PDK1 was correlated with poor prognosis in patients with NPC. PDK1 depletion enhanced radiosensitivity of NPC cells both in vitro and in vivo. Additionally, a specific PDK1 inhibitor also had the potential to enhance radiosensitivity in radioresistant NPC cells. Mechanistically, PDK1 depletion inhibited various targets of AKT including mTOR and GSK-3β and reversed the epithelial-mesenchymal transition. These findings indicated that PDK1 might be a potential target for NPC.

Knockdown of Annexin A2 Enhances Radiosensitivity by Increasing G2/M-Phase Arrest, Apoptosis and Activating the p38 MAPK-HSP27 Pathway in Nasopharyngeal Carcinoma.

Annexin A2 (ANXA2) has been found to be involved in cancer proliferation, metastasis and prognosis; however, its exact role in nasopharyngeal carcinoma (NPC) radioresistance remains unknown. We found that ANXA2 expression was correlated with prognosis in NPC patients, and longer overall survival in NPC patients with low ANXA2 expression than those with high ANXA2 expression. ANXA2 knockdown increased the radiosensitivity in radioresistant NPC cells, and ANXA2 overexpression decreased the radiosensitivity in NPC cells. Knocking-down ANXA2 expression increased the irradiation-induced apoptosis of radioresistant NPC cells, and ANXA2 overexpression decreased the irradiation-induced apoptosis of NPC cells. ANXA2 knockdown induced G2/M phase arrest in NPC cells post-irradiation, and ANXA2 overexpression abrogated G2/M phase arrest in NPC cells post-irradiation. ANXA2 overexpression resulted in inhibition of the p38 MAPK-HSP27 pathway, while ANXA2 knockdown resulted in activation of the p38 MAPK-HSP27 pathway. In addition, ANXA2 knockdown increased the radiosensitivity of the xenografted tumors in nude mice. Our data demonstrate that knockdown of Annexin A2 enhanced radiosensitivity in NPC by increasing G2/M-phase arrest, apoptosis and activating the p38 MAPK-HSP27 pathway. ANXA2 may be a promising target used to overcome radioresistance in NPC.

EGLN1 induces tumorigenesis and radioresistance in nasopharyngeal carcinoma by promoting ubiquitination of p53 in a hydroxylase-dependent manner.

Egl-9 Family Hypoxia Inducible Factor 1 (EGLN1) is a proline hydroxylase mediating degradation of hypoxia-inducible factor α (HIFα) through the ubiquitination system. Studies have indicated an essential role for EGLN1 in angiogenesis and tumorigenesis. However, there is no consensus on the regulation of EGLN1 and its mechanism of action on nasopharyngeal carcinoma (NPC). This study explored the association of the expression of EGLN1 with characteristics of NPC tumors and its underlying mechanism. We found that the expression of EGLN1 showed a positive correlation with tumor T classification and clinical staging of patients with NPC. EGLN1 could promote cell proliferation, invasion and migration, and even enhance the cancer stem cells (CSCs) prosperity and radioresistance of NPC cells. Mechanistically, EGLN1 facilitated degradation of tumor protein p53 through the ubiquitination system. This effect could be weakened in the presence of dimethyloxalylglycine (DMOG), suggesting that EGLN1 down-regulated p53 based on its hydroxylase activity. In conclusion, overexpression of EGLN1 promoted oncogenesis and induced a CSC-like phenotype in NPC cells, then enhancing the ability for radioresistance by interacting with p53 in a hydroxylase-dependent manner. Thus, EGLN1 might serve as a potential therapeutic target for NPC.

Radiosensitizer EXO-miR-197-3p Inhibits Nasopharyngeal Carcinoma Progression and Radioresistance by Regulating the AKT/mTOR Axis and HSPA5-mediated Autophagy

The biological functions of exosomes and microRNAs (miRs) in nasopharyngeal carcinoma (NPC) remain largely unexplored. Here, miR-197-3p was screened and identified, and whose level was reduced in serum and exosomes of patients with NPC. MiR-197-3p might be a good diagnostic and prognostic indicator. Our data showed that miR-197-3p expression was closely related to radioresistance, apoptosis, proliferation, migration, and survival of NPC. Inhibition of miR-197-3p expression in vitro could promote the proliferation and migration of NPC cells, while promotion of miR-197-3p expression in vivo could significantly inhibit the growth and enhance the radiosensitivity of NPC cells. From the perspective of mechanism, miR-197-3p could inhibit AKT/mTOR phosphorylation activation, inhibit an activated pathway of AKT/mTOR, target Heat Shock 70-kDa Protein 5(HSPA5) related to endoplasmic reticulum homeostasis, inhibit HSPA5-mediated autophagy, and reverse the radioresistance of NPC. Interestingly, exosomal miR-197-3p (EXO-miR-197-3p) reduced the proliferation and migration potential of NPC cells in vitro, and tumor growth and radioresistance of NPC cells in vivo. EXO-miR-197-3p inhibited NPC progression and radioresistance by regulating AKT/mTOR phosphorylation activation and HSPA5-mediated autophagy. In conclusion, our results highlight the potential of EXO-miR-197-3p as an effective radiosensitizer and therapeutic agent for refractory NPC.

Icotinib Enhanced Radiosensitization of Nasopharyngeal Carcinoma by Inhibiting the Expression of Epidermal Growth Factor Receptor

Epidermal growth factor receptor (EGFR) is frequently overexpressed in multiple malignancies. Icotinib (IH), a new EGFR tyrosine kinase inhibitor, enhances radiosensitivity in various types of cancer, but its effect on nasopharyngeal carcinoma (NPC) remains unclear. Total 115 NPC tissue sections and 30 nasopharyngitis tissue sections were enrolled. The correlation of EGFR expression and clinicopathologic features of NPC was analyzed. Survival analysis was calculated by using univariate and multivariate regression analysis. A radioresistant NPC cell line, CNE-2R, was established with a gradient irradiation schedule. Cell viability, colony formation and EGFR expression of CNE-2/2R cells were examined. Significant higher expression of EGFR was observed in NPC tissues than chronic nasopharyngitis lesions. EGFR expression was significantly correlated with both tumor stage (P < 0.001) and tumor-node-metastasis stage of NPC (P = 0.006). EGFR expression was an independent prognostic factor of disease-free survival (P = 0.047) and the overall survival of NPC (P = 0.016). Cell viability was higher in CNE-2R than CNE-2 on days 1, 2, 4, and 6 after radiation of 4 Gy. The colony number of CNE-2R was significantly higher than that of CNE-2 (P < 0.05), while IH enhanced the radiosensitizing effect of CNE-2R with lower survival fraction (P < 0.05). EGFR mRNA and protein expression levels were significantly higher in CNE-2R cells compared to CNE-2 cells, but significantly decreased after IH treatment (all P < 0.05). In conclusion, high EGFR expression is a poor prognostic factor for NPC patients. IH enhances the radiosensitivity of CNE-2R cells and reduce EGFR expression.

CRISPR/Cas9 genome-wide screening identifies LUC7L2 that promotes radioresistance via autophagy in nasopharyngeal carcinoma cells

Radioresistance emerges as the major obstacle to nasopharyngeal carcinoma (NPC) treatment, further understanding of underlying mechanisms is necessary to overcome the radioresistance and improve the therapeutic effect. In this study, we first identified a candidate radioresistant-related gene LUC7L2 via CRISPR/Cas9 high-throughput screening and quantitative proteomic approach. Overexpression of LUC7L2 in NPC cells promoted cell viability following exposure to ionizing radiation (IR), while knockdown of LUC7L2 significantly slowed down the DNA replication and impaired cell survival, sensitized NPC-radioresistant cells to IR. Using immunoprecipitation assay, we found SQSTM1, an autophagy receptor, was a potential binding partner of LUC7L2. Down-regulation of LUC7L2 in NPC-radioresistant cells led to reduction of SQSTM1 expression and enhancement of autophagy level. Furthermore, LUC7L2 knockdown in combination with autophagy inhibitor, chloroquine (CQ), resulted in more NPC-radioresistant cell death. Besides, LUC7L2 was obviously distributed in NPC tissues, and high LUC7L2 expression correlated with shorter survival in NPC patients. Our data suggest that LUC7L2 plays a huge part in regulating radioresistance of NPC cells, and serves as a promising therapeutic target in re-sensitizing NPC to radiotherapy.