Nasopharyngeal Carcinoma Stem Cells Research Articles

Inhibitory mechanism of KIF3A gene on nasopharyngeal carcinoma stem cells.

To investigate the inhibitory mechanism of the KIF3A gene on nasopharyngeal carcinoma (NPC) tumor stem cells. Set up a blank control group (BCG), NPC group, and KIF3A silence (si-KIF3A) group. The BCG cells were nasopharyngeal normal epithelial cell line NP69, without any treatment, and were cultured routinely; The NPC group cells are human NPC cell line CNE2 cells, which are not subjected to any treatment and are cultured routinely; si-KIF3A group cells were cultured in the offspring of human NPC cell line CNE2 infected with Lentivirus knockdown KIF3A gene. CCK8 was used to detect the cell proliferation ability, Western blot and qPCR were used to detect protein and related mRNA expression, while cell migration and invasion were detected using Transwell. The KIF3A protein and mRNA in the NPC group were higher than those in the BCG (P<0.05), while those in the si-KIF3A group cells were lower compared to BCG cells (P<0.05). The cell proliferation, migration and invasion activity in the si-KIF3A group were reduced than those in BCG (P<0.05). Si-KIF3A group cells HIF-1, NF-κB was reduced than that of BCG (P<0.05). The expression level of HIF-1, NF-κB protein in si-KIF3A group cells was reduced compared to BCG cells (P<0.05). Knocking down the KIF3A gene can reduce the vitality of NPC stem cells, and inhibit the malignant phenotype of NPC stem cells, via inhibiting HIF-1 and NF-κB expression.

MiR-138-1-3p alters the stemness and radiosensitivity of tumor cells by targeting CRIPTO and the JAK2/STAT3 pathway in nasopharyngeal carcinoma.

BackgroundTumor resistance to radiotherapy is one of the main obstacles to the clinical treatment of nasopharyngeal carcinoma (NPC). Improving the radiosensitivity of tumor cells has an important clinical significance in treatment of clinical NPC. This study aimed to identify that miR-138-1-3p as a novel therapeutic target in radioresistant NPC cells and found its targets, CRIPTO and the JAK2/STAT3 pathway.MethodsRadioresistant C666-IR and HK-1R cells were derived from the NPC cell lines C666-1 and HK-1. The different microRNAs (miRNAs) and their targeting genes were analyzed between C666-1 and C666-IR cells using microarray bioinformatics. Western blot, qRT-PCR, gene transfection, Luciferase reporter assay, and confocal laser scanning microscopy were applied for the analysis of the different genes.ResultsMiR-138-1-3p was found to target CRIPTO, which involved in the epithelial-mesenchymal transition (EMT) and JAK2/STAT3 signaling pathways. The luciferase reporter assay confirmed that miR-138-1-3p targeted CRIPTO and downregulated the expression of CRIPTO. Furthermore, miR-138-1-3p affected the stability of the CRIPTO-GRP78 complex on the cell membrane and also reversed the radioresistant characteristics of NPC stem cells, which affected EMT and the JAK2/STAT3 signaling pathway.ConclusionsThe miR-138-1-3p is a small molecule that can modulate radiosensitivity in the radioresistant C666-IR and HK-1R NPC cell lines by inhibiting EMT and targeting CRIPTO to reduce the activation of the JAK2/STAT3 pathway.

CD44 regulates biological behavior and Ras signaling pathway in nasopharyngeal carcinoma stem cells

Objective: To study the biological behavior of nasopharyngeal carcinoma stem cells and to explore the activation of Ras signaling pathway regulated by CD44. Methods: CNE2-SC and 5-8F-SC were nasopharyngeal carcinoma stem cells and obtained by serum-free suspension culture. Cell counting kit-8 (CCK-8) assay, colony formation assay, Transwell migration assay, cell adhesion array were used to investigate the growth, proliferation, migration and adhesion of nasopharyngeal carcinoma stem cells. Western blot test was used to detect the expressions of Ras signaling pathway related proteins and siRNA-mediated interference was used to determine the activation of Ras signaling pathway regulated by CD44. Results: The growth rates of CNE2-SC and 5-8F-SC cells were significantly lower than those of nasopharyngeal carcinoma cells at 24, 48 and 72 hours after inoculation (P<0.05). After 14 days of implantation, the colony formation rates of CNE2-SC (44.5±1.9)% and 5-8F-SC (47.4±1.8)% were higher than those of CNE2 (34.9±1.5)% and 5-8F (37.2±1.7)%, respectively(P<0.01). The migration cell number of CNE2-SC was (87.6±7.8), 3.97 times higher than that of CNE2 (P<0.01). The migration cell number of 5-8F-SC was (67.2±5.7), 3.07 times higher than 5-8F (P<0.01). The adhesion rates of CNE2-SC and CNE2 cells were (42.1±7.6)% and (8.9±2.0)%, respectively at 3 hours after inoculation and were (82.4±5.0)% and (12.1±2.2)% at 6 hours after inoculation, respectively. The adhesion rate of CNE2-SC cells was higher than that of CNE2 cells (all P<0.01). The adhesion rates of 5-8F-SC and 5-8F cells were (53.6±6.1)% and (7.3±1.5)% at 3 hours after inoculation, and (90.7±3.6)% and (11.0±1.2)% at 6 hours after inoculation, respectively. The adhesion rate of 5-8F-SC cells was higher than that of 5-8F cells (P<0.01). The expression levels of CD44, Ras and N-cadherin were significantly higher, while phosphatase and tensin homolog deleted on chromosome 10 (PTEN), E-cadherin in nasopharyngeal carcinoma stem cells were lower than those of the nasopharyngeal carcinoma cells. Furthermore, the levels of phosphorylated mitogen extracellular kinase1/2 (p-MEK1/2) and phosphorylated extracellular signal-regulated protein kinase1/2 (p-ERK1/2)were significantly increased in nasopharyngeal carcinoma stem cells (P<0.01). Correlation analysis showed that the protein expression levels of CD44 was highly positively correlated with RAS in nasopharyngeal carcinoma stem cells(r=0.985, P=0.002; r=0.962, P=0.038). Deletion of CD44 in CNE2-SC decreased the expression levels of HER-2, Ras and p-ERK1/2, p-Akt and phosphorylated protein kinase C-δ(p-PKCδ) (P<0.01). Conclusions: Despite compare to the nasopharyngeal carcinoma cell, nasopharyngeal carcinoma stem cells grows at a relatively slow rate, the capacities of clone formation, migration, adhesion are promoted. This may be related to the CD44-regulated abnormal activation of Ras signaling pathway.

Yi Qi Jie Du Formula and Salinomycin Combination Treatment Mediates Nasopharyngeal Carcinoma Stem Cell Proliferation, Migration and Apoptosis via CD44/Ras Signaling Pathway

ObjectiveTo assess the effects of Yi Qi Jie Du Formula (YQJDF) combined with salinomycin (SAL) on nasopharyngeal carcinoma stem cells (NPC-SCs) and investigate the underlying molecular mechanisms. MethodsCell counting methods, the CCK-8 assay, transwell migration assay and JC-1 staining, were used to observe the effects of the combination on the proliferation, migration and apoptosis of NPC stem cells, respectively. Western blot was used to detect the levels of protein in NPC-SCs. ResultsYQJDF combined with SAL had a synergistic effect on the inhibition of proliferation and migration and induction of NPC-SC apoptosis. Mechanistically, YQJDF combined with SAL synergistically upregulated the levels of apoptotic proteins, including cleaved Caspase-3, cleaved Caspase-7 and cleaved Caspase-9. Moreover, YQJDF combined with SAL synergistically decreased the levels of CD44, p-c-Src, Ras, p-PKCδ, p-MEK, p-c-Raf, p-ERK1/2 and p-AKT proteins. ConclusionsThe combination of YQJDF and SAL has a synergistic effect on the inhibition of NPC-SC proliferation and migration and induction of apoptosis, which may be closely related to the downregulation of the CD44/Ras signaling pathway.

Itraconazole attenuates the stemness of nasopharyngeal carcinoma cells via triggering ferroptosis.

Radiotherapy is a common therapy method for nasopharyngeal carcinoma (NPC) treatment; however, radioresistance greatly limits the clinical efficiency and prognosis of NPC patients. Therefore, it is extremely urgent to reveal the underlying mechanism contributing to radioresistance and find possible diagnostic biomarkers. Here, we collected the spheroids formed by NPC cells, which had been confirmed to hold the stem cell-like traits, and found that these spheroids exhibited a certain degree of radioresistance. Additionally, NPC spheroids displayed a certain degree of ferroptosis resistance, as evident by the decrease of iron concentration in lysosomes and lipid peroxides oxygen, and increase of glutathione (GSH) level. Furthermore, we revealed that itraconazole triggered the ferroptosis of NPC spheroids, which is characterized as the increase of iron concentration and lipid peroxides oxygen, and decrease of GSH level, and decreased the cell viability of NPC spheroids. Notably, itraconazole partially reversed the radioresistance of NPC spheroids. Mechanistically, we found that itraconazole can sequester iron in lysosome and thus trigger ferroptosis; this is essential for itraconazole-mediated attenuation on NPC spheroid stemness. Therefore, this study provides evidences showing that itraconazole might be used for killing NPC stem cells and thus attenuate radioresistance.

Lgr5 maintains stemness and regulates cell property in nasopharyngeal carcinoma through Wnt/β-catenin signaling pathway.

Nasopharyngeal carcinoma (NPC) is a common malignant tumor in Southern China and Southeast Asia. In this study, we found that Leucine rich repeat containing G protein-coupled receptor 5 (Lgr5) was highly expressed in NPC tissues and marked NPC stem cells. Lgr5high tumors showed differential transcriptional landscape compared to Lgr5not high tumors. Lgr5 expression was associated with the clinicopathologic features in NPC and was able to regulate the stemness and viability of NPC cell line CNE1 and HNE1. Meanwhile, the migration, invasion and epithelial-mesenchymal transition (EMT) was modulated by Lgr5 via Wnt/β-catenin signaling pathway. Furthermore, Lgr5 could regulate the sensitivity of NPC cells to chemotherapy drugs. Xenografted tumors from Lgr5-overexpressed CNE1 cells showed stronger tumor forming capacity and higher expression level of stem cell markers. Thus, we characterized previously unidentified role of Lgr5 in NPC cells, potential serving as a NPC stem cell biomarker and a therapeutic target against NPC.

The tumor-suppressive role of microRNA-873 in nasopharyngeal carcinoma correlates with downregulation of ZIC2 and inhibition of AKT signaling pathway.

Cancer stem cells (CSCs) are responsible for tumor initiation, relapse, and metastasis. Thus, residual CSCs after chemotherapy may result in poor prognosis for nasopharyngeal carcinoma (NPC). Emerging evidence suggests that differentially expressed microRNAs (miRNAs) regulate genes that carry out important functions in CSCs. Here we investigate the interaction of microRNA-873 (miR-873) with the Zic family member 2 (ZIC2) and the effects on downstream serine-threonine protein kinase (AKT) signaling pathway in CSCs in the context of NPC. Initially, microarray-based gene expression profiling identified ZIC2 as a key differentially expressed gene in NPC, which was subsequently confirmed to be upregulated in clinical NPC tissue samples. NPC cells were subjected to sphere-formation conditions in low-attachment plates, followed by sorting of CD133+ cells, which were selected as NPC stem cells after further characterization of stem cell biomarkers. ZIC2 was then shown to be enriched in NPC stem cells at both mRNA and protein levels. However, loss of ZIC2 was associated with the self-renewal, proliferative and tumorigenic properties of NPC stem cells. Next, miRNAs potentially able to target ZIC2 were predicted by the intersection of mirDIP and TargetScan database results, and miRNA miR-873 was found to be downregulated in NPC tissues in general but especially in NPC stem cells. Upregulation of miR-873 inhibited the stem-like properties and tumorigenicity of NPC stem cells, which was found to take place through downregulation of ZIC2 and disruption of the AKT signaling pathway. Collectively, the results obtained suggest that overexpression of miR-873 could aid NPC tumor suppression through reduction of the malignant potential of CSCs.

Association between Dose and Duration of Cisplatin Exposure with Sitotoksisity Effect on Nasopharyngeal Carcinoma Stem Cell

Background: Nasopharyngeal carcinoma (NPC) is ranked 6th of malignant tumors in Indonesia, while cisplatin is an effective chemotherapy therapy but the side effects and resistance problems are two major constraints limiting its application. Objective: To analyze the correlation of dose and duration of cisplatin exposure with cytotoxic effects on nasopharyngeal carcinoma stem cells Methods: A true experimental laboratory in vitro with the factorial design was used in this study. The biopsy NPC tissue was cultured and processed to obtain NPC stem cells to be treated with cisplatin exposure at different doses (0.05, 0.1, 0.2, 0.4, 0.8, 1 and 2 ?g) and different durations (24 and 48 hours). The number of dead cells after exposure will be calculated using a hemocytometer after 24, 48, and 72 hours of NPC stem cells free of cisplatin. Results: Death stem cell density of NPC was mostly obtained at the exposure of 2 ?g/ml cisplatin dose after 24 hours observation was 81.37%, while the smallest death cell density a dose of 0.05 ?g/ml after a 72-hour observation was 21.3%. Statistical analysis with multiple regression correlation tests between the density of death cell and cisplatin dose was obtained the coefficient correlation 0,827 and value p = 0,000. The analysis of the correlation between cisplatin exposure duration and death cell was also significant with the correlation coefficient -0.357 and the value p = 0.001. The cutoff point that correlated the dose and death stem cell of NPC by 50% in both 24 and 48 hours of exposure was 1 ?g (EC50). Conclusion: There was a correlation between the increased dose of cisplatin with the cytotoxicity effects on NPC stem cell.

Yi Qi Jie Du Decoction Inhibits Proliferation and Induces Apoptosis of Nasopharyngeal Carcinoma Stem Cells Through Mitochondrial Apoptosis Pathway

ObjectiveTo assess the effects of Yi Qi Jie Du Decoction (YQJDD) on nasopharyngeal carcinoma stem cells (NPC-SCs) and investigate the underlying mechanism. MethodsNPC-SCs were collected in serum-free culture system and identified by the sphere formation assay. The effect of YQJDD on the proliferation of NPC-SCs was detected using the Cell Counting Kit-8 assay. The change of mitochondrial membrane potential (ΔΨm) in NPC-SCs following treatment with YQJDD was investigated by JC-1 staining. The levels of apoptosis- associated proteins were examined using western blot analysis. ResultsYQJDD significantly inhibited the proliferation of NPC-SCs in dose- and time-dependent manners. JC-1 staining revealed that YQJDD lowered the ΔΨm of NPC-SCs in a time- dependent manner. After treatment with YQJDD, the expression of cleaved caspase-3, -7 and -9, cleaved poly-ADP ribose polymerase, P21 and P53 were increased, while the expression of Survivin in NPC-SCs was decreased. However, the expression of cleaved caspase-8 remained nearly unchanged. ConclusionsYQJDD inhibits the growth and induces apoptosis of NPC-SCs via an intrinsic apoptosis pathway.

Casticin inhibits nasopharyngeal carcinoma growth by targeting phosphoinositide 3-kinase

BackgroundCasticin, an isoflavone compound extracted from the herb Fructus Viticis, has demonstrated anti-inflammatory and anticancer activities and properties. The aim of this study was to investigate the effects and mechanisms of casticin in nasopharyngeal carcinoma (NPC) cells and to determine its potential for targeted use as a medicine.MethodsNPC cells were used to perform the experiments. The CCK‑8 assay and colony formation assays were used to assess cell viability. Flow cytometry was used to measure the cell cycle and apoptosis analysis (annexin V/PI assay). A three-dimensional (3D) tumour sphere culture system was used to characterize the effect of casticin on NPC stem cells. In silico molecular docking prediction and high-throughput KINOME scan assays were used to evaluate the binding of casticin to phosphoinositide 3-kinase (PI3K), including wild-type and most of mutants variants. We also used the SelectScreen assay to detect the IC50 of ATP activity in the active site of the target kinase. Western blotting was used to evaluate the changes in key proteins involved cell cycle, apoptosis, stemness, and PI3K/protein kinase B (AKT) signalling. The effect of casticin treatment in vivo was determined by using a xenograft mouse model.ResultsOur results indicate that casticin is a new and novel selective PI3K inhibitor that can significantly inhibit NPC proliferation and that it induces G2/GM arrest and apoptosis by upregulating Bax/BCL2 expression. Moreover, casticin was observed to affect the self-renewal ability of the nasopharyngeal carcinoma cell lines, and a combination of casticin with BYL719 was observed to induce a decrease in the level of the phosphorylation of mTORC1 downstream targets in BYL719-insensitive NPC cell lines.ConclusionCasticin is a newly emerging selective PI3K inhibitor with potential for use as a targeted therapeutic treatment for nasopharyngeal carcinoma. Accordingly, casticin might represent a novel and effective agent against NPC and likely has high potential for combined use with pharmacological agents targeting PI3K/AKT.

Cinobufotalin Powerfully Reversed EBV-miR-BART22-Induced Cisplatin Resistance via Stimulating MAP2K4 to Antagonize Non-Muscle Myosin Heavy Chain IIA/Glycogen Synthase 3β/β-Catenin Signaling Pathway

Background: Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-related tumor. The role of EBV-encoding miR-BART-22 is still unclear in NPC. This study aimed to identify the detailed mechanisms by which EBV-miR-BART22 functions as a tumor-promoting factor and evaluate the action of cinobufotalin in treating EBV-miR-BART22-overexpressing NPC cells. Methods: Flow cytometry was used to identify the EBV-miR-BART22- overexpressing NPC stem cells. Sphere formation assay, western blotting, qRT-PCR assay, transwell, boyden, xenograft assay and immunohistochemistry staining analysis were utilized to evaluate the effect of EBV-miR-BART22 on the cells metastasis and DDP chemoresistance. qRT-PCR assay, immunofluorescence staining, Immunofluorescence, luciferase reporter assay, CHIP, EMSA and Co-IP assay were performed to explore the detailed molecular mechanism of EBV-miR-BART22 and its target gene. Findings: We observed that EBV-miR-BART22 not only promoted tumor stemness and metastasis, but also enhanced the resistance to Cisplatin (DDP) in vitro and in vivo. Mechanistic analyses indicated that EBV-miRBART-22 directly targeted the MAP2K4 and upregulated non-muscle myosin heavy chain IIA (MYH9) expression by PI3K/AKT/c-Jun-induced transcription. Further, MYH9 interacted with glycogen synthase 3β(GSK3β) protein and induced its ubiquitin degradation by activating PI3K/AKT/cJun-induced ubiquitin transcription and the latter combined with increased TRAF6 E3 ligase, which further bound to GSK3β protein. Reductions in the GSK3β protein thus promoted β-catenin expression and nuclear translocation, which induced tumor stemness and the epithelialto-mesenchymal transition (EMT) signals. Furthermore, we observed that cinobufotalin, a new chemically synthesized compound, significantly suppressed EBV-miR-BART-22-induced DDP chemoresistance by upregulating MAP2K4 to suppress MYH9/GSK3β/β-catenin and its downstream tumor stemness and EMT signals in NPC. Finally, clinical data revealed that increased miR-BART-22 and reduced MAP2K4 expression caused the poor prognoses of NPC patients. Interpretation: Our study provides a novel mechanism that cinobufotalin reversed the DDP chemoresistance and EMT induced by EBV-miR-BART22 in NPC. Funding Statement: China (No.81572643,81772872), People's Livelihood Science and technology grant of Guangzhou Municipal Science and technology project(No. 201803010023), Guangzhou science and technology plan(No. 201804010023), The Supporting plan for Special Talents in Guangdong Province (No. 2016TQ03R466), Project of Guangdong Province (No.2016A020215233), National Science Foundation for Young Scientists of China (No.81702672). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: For the use of the clinical materials for research purpose, approval from the Ethics Committee of the Nanfang Hospital were obtained. All animal experiments were conducted in accordance with a protocol approved by the Animal Care and Use Committee of Integrated Hospital of Traditional Chinese Medicine of Southern Medical University.

Effects of D-α-tocopherol polyethylene glycol succinate-emulsified poly(lactic-co-glycolic acid) nanoparticles on the absorption, pharmacokinetics, and pharmacodynamics of salinomycin sodium.

Although salinomycin sodium (SS) has shown in-vitro potential to inhibit cancer stem cell growth and development, its low water solubility makes it a poor candidate as an oral chemotherapeutic agent. To improve the bioavailability of SS, SS was encapsulated here using D-α-tocopherol polyethylene glycol succinate (TPGS)-emulsified poly(lactic-co-glycolic acid) (PLGA) nanoparticles and compared with its parent SS in terms of absorption, pharmacokinetics, and efficacy in suppressing nasopharyngeal carcinomas stem cells. The pharmacokinetics of SS and salinomycin sodium-loaded D-α-tocopherol polyethylene glycol succinate-emulsified poly(lactic-co-glycolic acid) nanoparticles (SLN) prepared by nanoprecipitation were analyzed in-vivo by timed-interval blood sampling and oral administration of SS and SLN to rats. Sensitive liquid chromatography-mass spectrometry (LC-MS) was developed to quantify plasma drug concentrations. SS and SLN transport in Caco-2 cells was also investigated. The therapeutic efficacy of SS and SLN against cancer stem cells was determined by orally administering the drugs to mice bearing CNE1 and CNE2 nasopharyngeal carcinoma xenografts and then evaluating CD133 cell proportions and tumorsphere formation. The in-vivo trial with rats showed that the Cmax, AUC(0-t), and Tmax for orally administered SLN were all significantly higher than those for SS (P<0.05). These findings were corroborated by a Caco-2 cell Transwell assay showing that relative SLN absorption was greater than that of SS on the basis of their apparent permeability coefficients (Papp). Significantly, therapeutic SLN efficacy against nasopharyngeal carcinoma stem cells was superior to that of SS. TPGS-emulsified PLGA nanoparticles effectively increase SS solubility and bioavailability. SLN is, therefore, promising as an oral chemotherapeutic agent against cancer stem cells.

Association Between Dose and Duration of Cisplatin Exposure with Cytotoxicity Effect on Nasopharyngeal Carcinoma Stem Cell.

Nasopharyngeal carcinoma (NPC) is ranked 6th of malignant tumors in Indonesia. To analyze the correlation of dose and duration of cisplatin exposure with cytotoxic effects on nasopharyngeal carcinoma stem cells. The biopsy NPC tissue was cultured and processed to obtain NPC stem cells to be treated with cisplatin different doses and durations (24 and 48h). The number of dead cells after exposure will be calculated using a hemocytometer. Death stem cell density of NPC at exposure of 2μg/ml cisplatin dose was 81.37%, while the smallest death cell density a dose of 0.05μg/ml after a 72-h observation was 21.3%. The coefficient correlation 0.827 and value p = 0.000 (p < 0.05). The analysis of the correlation between cisplatin exposure duration and death cell was also significant with the correlation coefficient -0.357 and the value p = 0.001 (p < 0.05). There was a correlation between the increased dose of cisplatin with the cytotoxicity effects on NPC stem cell.

XIAP Limits Autophagic Degradation of Sox2 and Is A Therapeutic Target in Nasopharyngeal Carcinoma Stem Cells.

Rationale: Nasopharyngeal carcinoma (NPC) is the most frequent head and neck tumor in South China. The presence of cancer stem cells (CSCs) in NPC contributes to tumor maintenance and therapeutic resistance, while the ability of CSCs to escape from the apoptosis pathway may render them the resistant property to the therapies. Inhibitor of apoptosis proteins family proteins (IAPs), which are overexpressed in nasopharyngeal carcinoma stem cells, may play an important role in maintaining nasopharyngeal cancer stem cell properties. Here, we develop a novel CSC-targeting strategy to treat NPC through inhibiting IAPs.Methods: Human NPC S-18 and S-26 cell lines were used as the model system in vitro and in vivo. Fluorescence activated cell sorting (FACS) assay was used to detect nasopharyngeal SP cells and CD44+ cells. The characteristics of CSCs were defined by sphere suspension culture, colony formation assay and cell migration. The role of XIAP on the regulation of Sox2 protein stability and ERK1-mediated phosphorylation of Sox2 signaling pathway were analyzed using immunoblotting, immunoprecipitation, immunofluorescence, phosphorylation mass spectrometry, siRNA silencing and plasmid overexpression. The correlation between XIAP and Sox2 in NPC biopsies and their role in prognosis was performed by immunohistochemistry. APG-1387 or chemotherapies-induced cell death and apoptosis in S-18 and S-26 were determined by WST, immunoblotting and flow cytometry assay.Results: IAPs, especially X chromosome-linked IAP (XIAP), were elevated in CSCs of NPC, and these proteins were critically involved in the maintenance of CSCs properties by enhancing the stability of Sox2. Mechanistically, ERK1 kinase promoted autophagic degradation of Sox2 via phosphorylation of Sox2 at Ser251 and further SUMOylation of Sox2 at Lys245 in non-CSCs. However, XIAP blocked autophagic degradation of Sox2 by inhibiting ERK1 activation in CSCs. Additionally, XIAP was positively correlated with Sox2 expression in NPC tissues, which were associated with NPC progression. Finally, we discovered that a novel antagonist of IAPs, APG-1387, exerted antitumor effect on CSCs. Also, the combination of APG-1387 with CDDP /5-FU has a synergistic effect on NPC.Conclusion: Our study highlights the importance of IAPs in the maintenance of CSCs in NPC. Thus, XIAP is a promising therapeutic target in CSCs and suggests that NPC patients may benefit from a combination treatment of APG-1387 with conventional chemotherapy.

5T4-specific chimeric antigen receptor modification promotes the immune efficacy of cytokine-induced killer cells against nasopharyngeal carcinoma stem cell-like cells

Relapse and metastasis of nasopharyngeal carcinoma (NPC) are presumably attributed to cancer stem cells (CSCs). In recent years, chimeric antigen receptor (CAR)-modified immune effector cells have been shown to have impressive antitumour efficacy. In this study, we aimed to identify appropriate tumour-associated antigens predominantly expressed on NPC stem cells (NPCSCs) and determine their suitability for CAR-engineered cytokine-induced killer (CIK) cell therapy against NPC. By investigating the expression patterns of potential target antigens (ROR1, 5T4 and CAIX) in NPC, we found that the oncofetal antigen 5T4 was predominately expressed in NPC cell lines and tissues but absent in non-cancerous nasopharyngeal tissues. Moreover, significantly enhanced expression of 5T4 in NPC spheroids revealed its relationship with putative NPCSCs. Hence, we designed a CAR construct (5T4-28Z) specific for 5T4 and generated CAR-transduced CIK cells. Our results showed that the artificial CAR was efficiently expressed on the surface of CIK cells and that no native phenotypes were altered by the gene transduction. Functional assays revealed that 5T4-28Z-CIK cells possessed both CAR-mediated and CAR-independent anti-NPC activity and were capable of efficiently attacking NPC cells, especially NPCSC-like cells in vitro, suggesting that they might serve as an attractive tool for developing efficient therapies against NPC.

Smac Mimetics in Combination with TRAIL Selectively Target Cancer Stem Cells in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma is a common malignancy in Southern China. After radiotherapy and chemotherapy, a considerable proportion of patients with nasopharyngeal carcinoma suffered tumor relapse and metastasis. Cancer stem cells (CSC) have been shown with resistance against therapies and thus considered as the initiator of recurrence and metastasis in tumors, where the antiapoptotic property of CSCs play an important role. Smac/DIABLO is an inverse regulator for the inhibitors of apoptosis protein family (IAP), which have been involved in apoptosis. Here, the effects of Smac mimetics on the CSCs of nasopharyngeal carcinoma were studied both in vitro and in vivo, using two clones of nasopharyngeal carcinoma cell line CNE2 as models. We found that one of the clones, S18, had CSC-like properties and IAPs were overexpressed. The combination of Smac mimetics and TNF-related apoptosis-inducing ligand (TRAIL) can reduce the percentage of SP cells and inhibit the colony- and sphere-forming abilities of S18 cells, indicating their ability to attenuate the CSCs. Moreover, in a nasopharyngeal carcinoma xenograft model, the administration of Smac mimetics in combination with TRAIL also led to the elimination of nasopharyngeal carcinoma stem cells. Furthermore, the Smac mimetics in combination with TRAIL induced the degradation of cIAP1 and XIAP and thus induced apoptosis in vitro and in vivo. Taken together, our data show that Smac mimetics exerted an antitumor effect on nasopharyngeal carcinoma cancer stem cells, and this combination treatment should be considered as a promising strategy for the treatment of nasopharyngeal carcinoma.