Cancer Stem Cells In Nasopharyngeal Carcinoma Research Articles

Protein C receptor maintains cancer stem cell properties via activating lipid synthesis in nasopharyngeal carcinoma

Metastasis and recurrence account for 95% of deaths from nasopharyngeal carcinoma (NPC). Cancer stem cells (CSCs) are regarded as one of the main reasons for tumor cell resistance to clinical therapy, and cancer metastasis or recurrence, while little is known about CSCs in NPC. The present study uncovers a subpopulation of cells labeled as CD45−EPCAM+PROCR+ in NPC biopsy samples that exhibit stem cell-like characteristics. A relatively low number of these cells initiate xenograft tumors in mice. Functional analysis reveals that protein C receptor (PROCR) not only serves as a stem cell marker in NPC, but also maintains tumor cells’ stemness potential through regulating lipid metabolism and mitochondrial fission. Epistatic studies reveal that cAMP-protein kinase A stimulates Ca2+ release to manipulate lipid metabolism related genes’ expression. Finally, in a cohort of 207 NPC samples, PROCR expression is correlated with tumor metastasis or recurrence, and predicts poor prognosis. These novel findings link PROCR labeled CSCs with lipid metabolism and mitochondrial plasticity, and provides new clinical target against metastatic or recurrent NPC.

Prognostic value of circulating tumor cells and its association with the expression of cancer stem cells in nasopharyngeal carcinoma patients

The release of circulating tumor cells (CTCs) into vasculature is an early event in the metastatic process and the detection of CTCs has been widely used clinically. In addition, cancer stem cells (CSCs) are the source of distant metastasis. However, the relationship between CTCs and CSCs in nasopharyngeal carcinoma (NPC) patients was largely unknown. A total of 93 NPC patients were enrolled in this study. The CTCs in the peripheral blood were detected. The expression of ALDH1A1 in the tumor tissues of the corresponding patients was detected using immunohistochemistry (IHC). The prognostic value of CTCs level and the correlation with the expression of ALDH1A1 was evaluated. Data showed that the detection of CTCs was positively correlated with metastasis (p<0.001). The positive detection of CTCs was also associated with poor overall survival (p=0.025). CTCs ≥2 demonstrated good specificity and sensitivity in predicting distant metastasis, while CTCs ≥8 demonstrated better specificity and sensitivity in predicting prognosis than CTCs ≥2. Furthermore, we found that there was a positive relationship between the detection of CTCs and the expression of ALDH1A1 (p=0.001). The prognosis analysis also demonstrated that high ALDH1A1 expression was correlated with poor overall survival (p=0.006). Our study demonstrated a positive correlation between the CTCs and the expression of CSCs, both were positively correlated with metastasis and poor prognosis. These results indicated that the CTCs might indirectly reflect the expression of CSCs.

MicroRNA-124 inhibits stem-like properties and enhances radiosensitivity in nasopharyngeal carcinoma cells via direct repression of expression of JAMA.

Cancer stem cells (CSCs) are a source of tumour recurrence in patients with nasopharyngeal carcinoma (NPC); however, the function of microRNA‐124 (miR‐124) in NPC CSCs has not been clearly defined. In this study, we investigated the role of miR‐124 in NPC CSCs. qRT‐PCR was performed to measure miR‐124 expression in NPC tissues and cell lines and the effects of miR‐124 on stem‐like properties and radiosensitivity of NPC cells measured. Luciferase reporter assays and rescue experiments were used to investigate the interaction of miR‐124 with the 3′UTR of junctional adhesion molecule A (JAMA). Finally, we examined the effects of miR‐124 in an animal model and clinical samples. Down‐regulation of miR‐124 was detected in cancer tissues and was inversely associated with tumour stage and lymph node metastasis. Overexpression of miR‐124 inhibited stemness properties and enhanced radiosensitivity of NPC cells in vitro and in vivo via targeting JAMA. Up‐regulation of miR‐124 was correlated with superior overall survival of patients with NPC. Our study demonstrates that miR‐124 can inhibit stem‐like properties and enhance radiosensitivity by directly targeting JAMA in NPC. These findings provide novel insights into the molecular mechanisms underlying therapy failure in NPC.

Latent Membrane Protein 1 Antibody Inhibits Cell Migration, Invasion and Epithelial-Mesenchymal Transition of Nasopharyngeal Carcinoma Stem Cell via Regulating Wnt/β-Catenin Signaling Pathway

Objective: In order to investigate the effects of LMP1-Fab antibody on Nasopharyngeal carcinoma (NPC) cancer stem cells (CSCs). Methods/ Results: Methods were performed to study the effects of LMP1-Fab antibody on NPC CSCs in vivo and in vitro, for example, transwell chamber assay, wound healing assay, western blot assay, quantitative real-time PCR assay animal experiments, animal fluorescence imaging, H&amp;E staining, immunohistochemistry. We identified that LMP1 activated the migration and invasion of NPC. Whereas the LMP1-Fab antibody inhibited cell invasion, epithelial-mesenchymal transition (EMT) and migration of NPC CSCs in LMP1+ HNE2 cells. Furthermore, LMP1-Fab antibody significantly increased the expression of E-cadherin, and reduced the expressions of vimentin,N -cadherin and Slug in LMP1+ HNE2 CSCs cells. Mechanistically, LMP1-Fab antibody inhibited lung and liver metastasis by regulating the wnt/ -catenin pathway in the nude mice. Conclusion: These results suggested that the novel antibody-targeting LMP1 is likely to be a potential strategy for the treatment of NPC.

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.

The developmental transcription factor IRF6 attenuates ABCG2 gene expression and distinctively reverses stemness phenotype in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC), which originates from the nasopharynx, is highly prevalent in Southern China and Southeast Asia, and more than 90% of all NPCs are non-keratinizing undifferentiated cells or poorly differentiated squamous cells. Cancer stem cells (CSCs) are capable of self-renewal and have differentiation potential. These properties form the basis of cancer initiation, development, and radiochemoresistance. However, the molecular mechanisms underlying NPC CSC maintenance remain poorly understood. Here, genomic expression profiling using our previously established monoclonal cellular and animal models revealed that interferon regulatory factor 6 (IRF6) was downregulated in highly metastatic NPC cells, cancer stem-like NPC cells and animal models. Functional assays revealed that elevated IRF6 expression suppressed cell proliferation, growth, CSCs properties and enhanced cell chemotherapeutic sensitivity. However, silencing IRF6 resulted in opposing effects. Moreover, we determined that as a tumor suppressor gene and transcription factor, IRF6 directly bound the upstream region of the ATP-binding cassette sub-family G member 2 (ABCG2) DNA element and suppressed target ABCG2 expression in NPC cells. Consistently, an inverse correlation was observed between the mRNA levels of IRF6 and ABCG2 in clinical NPC samples. With these results, we provide the first evidence that IRF6 directly targets the ABCG2 gene and selectively kills CSCs in NPC and that IRF6 may be a valuable tool for developing new CSC-targeted treatment strategies for undifferentiated NPC patients.

NOR1 Suppresses Cancer Stem-Like Cells Properties of Tumor Cells via the Inhibition of the AKT-GSK-3β-Wnt/β-catenin-ALDH1A1 Signal Circuit.

Cancer stem cells (CSCs) play a key role in tumor radiotherapy and chemotherapy resistance, relapse, and metastasis, and are primarily maintained in a resting state in vivo. The failure of conventional therapies to target CSCs is the main cause of treatment failure. The discovery of CSCs in nasopharyngeal carcinoma (NPC) tumors is becoming more prevalent; however, the understanding of the mechanisms underlying the maintenance of tumor stemness is still limited. We previously cloned NOR1, a tumor suppressor gene downregulated in NPC cell lines and tissues. In this study, we demonstrate that Wnt/β-catenin and ALDH1A1 form a signal circuit and that NOR1 antagonizes the tumor stem cell-like phenotype in NPC cell lines: the ectopic overexpression of NOR1 reduced β-catenin and ALDH1A1 expression; β-catenin/TCF4 targeted the regulation of ALDH1A1 transcription in NPC cells; silencing ALDH1A1 reduced AKT (total and phosphorylated) and GSK-3β (phosphorylated) expression; and eventually feedback decreased β-catenin expression levels. We also found that NOR1 expression decreased cancer stem-like cell properties of NPC cells, reduced their ability to form tumor spheroids in vitro, reduced tumorigenicity in nude mice in vivo, and increased sensitivity to chemotherapy agents. Taken together, our findings illustrated a new function of NOR1 that suppresses cancer stem-like cell properties in tumor cells by inhibiting the AKT-GSK-3β-Wnt/β-catenin-ALDH1A1 signal circuit. The study suggests that NOR1 deletion expression in NPC cells may be a potential molecular target for cancer stem cell therapy. J. Cell. Physiol. 232: 2829-2840, 2017. © 2016 Wiley Periodicals, Inc.

Lovastatin Inhibits Cancer Stem Cells and Sensitizes to Chemo- and Photodynamic Therapy in Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus-associated malignancy occurring at high incidence in Southeast Asia and southern China. In spite of the good response to radio- and chemo-therapy at the early stage, resistance and recurrence develop in NPC patients in the advanced setting. Cancer stem cells (CSCs) play an important role in drug resistance and cancer recurrence. Here we report that lovastatin, a natural compound and a lipophilic statin that has already been used in the clinic to treat hypercholesterolemia, inhibited the CSC properties and induced apoptosis and cell cycle arrest in sphere-forming cells derived from the 5-8F and 6-10B NPC cell lines. Furthermore, lovastatin conferred enhanced sensitivity to the chemotherapeutic and photodynamic agents in NPC CSCs. Together our findings suggest that targeting CSCs by lovastatin in combination with routine chemotherapeutic drugs or photodynamic therapy might be a promising approach to the treatment of NPC.

Downregulation of mitochondrial cyclooxygenase-2 inhibits the stemness of nasopharyngeal carcinoma by decreasing the activity of dynamin-related-protein 1

Cancer stem cells (CSCs) are a small subset of malignant cells, possessing stemness, with strong tumorigenic capability, conferring resistance to therapy and leading to the relapse of nasopharyngeal carcinoma (NPC). Our previous study suggested that cyclooxygenase-2 (COX-2) would be a novel target for the CSCs-like side population (SP) cells in NPC. In the present study, we further found that COX-2 maintained the stemness of NPC by enhancing the activity of mitochondrial dynamin-related protein 1 (Drp1), a mitochondrial fission mediator, by studying both sorted SP cells from NPC cell lines and gene expression analyses in NPC tissues. Using both overexpression and knockdown of COX-2, we demonstrated that the localization of COX-2 at mitochondria promotes the stemness of NPC by recruiting the mitochondrial translocation of p53, increasing the activity of Drp1 and inducing mitochondrial fisson. Inhibition of the expression or the activity of Drp1 by siRNA or Mdivi-1 downregulates the stemness of NPC. The present study also found that inhibition of mitochondrial COX-2 with resveratrol (RSV), a natural phytochemical, increased the sensitivity of NPC to 5-fluorouracil (5-FU), a classical chemotherapy drug for NPC. The underlying mechanism is that RSV suppresses mitochondrial COX-2, thereby reducing NPC stemness by inhibiting Drp1 activity as demonstrated in both the in vitro and the in vivo studies. Taken together, the results of this study suggest that mitochondrial COX-2 is a potential theranostic target for the CSCs in NPC. Inhibition of mitochondrial COX-2 could be an attractive therapeutic option for the effective clinical treatment of therapy-resistant NPC.

Abstract 3360: EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngealcarcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop

Abstract Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44+/High stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition (EMT)-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered PI3K/AKT pathway, which subsequently stimulated expression of CSC markers, development of side population (SP) and tumor sphere formation. This suggests that PI3K/AKT pathway plays an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44+/High cells. In addition, LMP1 greatly increased expression of miR-21 and down-regulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by a miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and up-regulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and play a key role in LMP1-induced CSCs in NPC. Citation Format: Chang-Fu Yang, Guang-Da Yang, Tie-Jun Huang, Jia-Ling Huang, Chao-Nan Qian, Bi-Jun Huang. EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngealcarcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3360.

EB-virus latent membrane protein 1 potentiates the stemness of nasopharyngeal carcinoma via preferential activation of PI3K/AKT pathway by a positive feedback loop.

Our previous study reported that Epstein-Barr virus(EBV)-encoded latent membrane protein 1 (LMP1) could induce development of CD44(+/High) stem-like cells in nasopharyngeal carcinoma (NPC). However, the molecular mechanisms that underlie modulation of cancer stem cells (CSCs) in NPC remain unclear. Here, we show that LMP1 induced CSC-like properties through promotion of the expression of epithelial-mesenchymal transition-like cellular markers and through alterations in differentiation markers. Furthermore, LMP1 activated and triggered phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway, which subsequently stimulated expression of CSC markers, development of side population and tumor sphere formation. This suggests that PI3K/AKT pathway has an important role in the induction and maintenance of CSC properties in NPC. Similarly, PI3K/AKT pathway was also activated by phosphorylase in LMP1-induced CD44(+/High) cells. In addition, LMP1 greatly increased expression of miR-21 and downregulated expression of the miR-21 target, PTEN. Overexpression of miR-21 by transfection of miR-21 mimics into LMP1-transformed cells led to phosphorylase-mediated activation of the PI3K/AKT pathway and induction of CSCs. On the contrary, phosphorylation of the PI3K/AKT pathway and the expression of CSC were reversed by an miR-21 inhibitor. The specific inhibitor (Ly294002) of PI3K/AKT pathway significantly decreased expression of miR-21 and CSC markers and upregulated the expression of PTEN, which indicates that miR-21 and PTEN are the downstream effectors of PI3K/AKT and that expression of these two effectors are related to the development of NPC CSCs. Taken together, our novel findings indicate that LMP1, PI3K/AKT, miR-21 and PTEN constitute a positive feedback loop and have a key role in LMP1-induced CSCs in NPC.

Abstract 1753: XIAP maintains the characteristics of cancer stem cells and is a therapeutic target in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is a common malignancy in South China. Cancer stem cells (CSCs) are thought as the key players in the resistance to chemo- or radio-therapy and are response for tumor recurrence and metastasis, while their ability to escape from the apoptosis pathway may render them the resistant property to the therapies. We show that XIAP is overexpressed in the NPC cell line S18, which have the higher SP populations and CD44+ populations and the stronger abilities of sphere-formation and metastasis. Importantly, XIAP knockdown decreases SP and CD44+ populations, prevents sphere-formation and reduces the ability of invasion and migration. What more, loss of XIAP enhances the sensitivity of cells to conventional chemotherapy drugs CDDP and 5-FU. The primary xenograft tumor formation in vivo found there is a dramatic reduction in tumor formation in the XIAP KD group. They all indicate that XIAP plays an important role in nasopharyngeal cancer stem cell. Further studies show that XIAP limits MAPK signaling and thereby restricts differentiation and maintains the characteristics of cancer stem cells. We designed and synthesized a novel Smac mimetic compound APG-1387 that promotes the rapid degradation of cIAP1/2 and XIAP, and it exerts an antitumor effect on nasopharyngeal carcinoma cancer stem cells. Further studies show that APG-1387 enhances the chemosensitivity and promotes apoptosis in combination with CDDP and 5-FU of NPC in vitro and vivo. Citation Format: jiao ji, Rong Deng, Wen-Dan Chen, Gong-Kan Feng, Xiao-Feng Zhu, Da-Jun Yang. XIAP maintains the characteristics of cancer stem cells and is a therapeutic target in nasopharyngeal carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1753. doi:10.1158/1538-7445.AM2015-1753

Downregulation of cancer stem cell properties via mTOR signaling pathway inhibition by rapamycin in nasopharyngeal carcinoma.

Rapamycin, a mammalian target of rapamycin (mTOR) signaling inhibitor, inhibits cancer cell proliferation and tumor formation, including in nasopharyngeal carcinoma (NPC), which we proved in a previous study. However, whether rapamycin affects cancer stem cells (CSCs) is unclear. In examining samples of NPCs, we found regions of CD44-positive cancer cells co-expressing the stem cell biomarker OCT4, suggesting the presence of CSCs. Following this, we used double-label immunohistochemistry to identify whether the mTOR signaling pathway was activated in CD44-positive CSCs in NPCs. We used a CCK-8 assay and western blotting to explore whether the stem cell biomarkers CD44 and SOX2 and the invasion protein MMP-2 could be suppressed by treatment with rapamycin in cultured primary NPC cells and secondary tumors in BALB/c nude mice. Interestingly, we found that rapamycin inhibited mTOR signaling in addition to simultaneously downregulating the expression of CD44, SOX2 and MMP-2 and that it affected cell growth and tumor size and weight both in vitro and in vivo. Collectively, we confirmed for the first time that CSC properties are reduced and invasion potential is restrained in response to mTOR signaling inhibition in NPC. This evidence indicates that the targeted inhibition of CSC properties may provide a novel strategy to treat cancer.

Cancer stem cells in nasopharyngeal carcinoma: current evidence

Nasopharyngeal carcinoma (NPC) is highly prevalent in southern China and Southeast Asia. Cancer resistance to therapy, metastasis and disease recurrence are significant hurdles to successful treatment of NPC. Identifying mechanisms by which NPC is resistant is critical to improving patient survival. Evidence gathered in the last decade suggests that tumor progression and recurrence may be fuelled by cancer stem cells (CSCs). Understanding how CSCs contribute to the pathology of NPC will potentially aid the pursuit of novel therapies. In this review we summarize what major methods are currently used to identify CSCs in NPC and the challenges faced.

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.

Nigericin selectively targets cancer stem cells in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is prevalent in southern China, northern Africa, and Alaska. The prognosis for NPC patients at early stage is good, while it is poor for patients at late stages. Cancer stem cells (CSCs) have been proposed to be associated with tumor initiation, relapse and metastasis, and the poor prognosis of NPC likely results from residual CSCs after therapy. Study on the therapy targeting CSCs in NPC remains poor, though it received intensive attentions in other cancers. Here, we used NPC cell lines with high and low proportion of CSCs as models to explore the effect of nigericin, an antibiotic, on CSCs. We found that nigericin could selectively target CSCs and sensitize CSCs in NPC to the widely used clinical drug cisplatin both in vitro and in vivo. Moreover, downregulation of the polycomb group protein Bmi-1 may contribute to the inhibitory effect of nigericin on CSCs. Furthermore, by using the in vitro NPC cell models, we found that nigericin could significantly decrease the migration and invasion abilities, which are known to be associated with CSCs. Taken together, our results suggest that nigericin can selectively target CSCs in NPC, which could be a candidate CSCs targeting drug for clinical evaluation.

Resveratrol Impedes the Stemness, Epithelial-Mesenchymal Transition, and Metabolic Reprogramming of Cancer Stem Cells in Nasopharyngeal Carcinoma through p53 Activation

Cancer stem cells (CSCs) are able to self-renew and are refractory to cancer treatment. To investigate the effects of resveratrol on CSCs of nasopharyngeal carcinoma (NPC), we employed a behavior selection strategy to isolate CSCs based on radioresistance, chemoresistance, and tumor sphere formation ability. These NPC CSCs displayed stem cell properties and underwent metabolic shift to predominately rely on glycolysis for energy supply. Intriguingly, we found that resveratrol turned off the metabolic switch, increased the reactive oxygen species (ROS) level, and depolarized mitochondrial membranes. These alterations in metabolism occurred concomitantly with the suppression of CSC properties including resistance to therapy, self-renewal capacity, tumor initiation capacity, and metastatic potential in NPC CSCs. We found that resveratrol impeded CSC properties through the activation of p53 and this effect could be reversed by knockdown of p53. Furthermore, resveratrol suppressed the stemness and EMT through reactivating p53 and inducing miR-145 and miR-200c, which were downregulated in NPC CSCs. In conclusion, we demonstrated that resveratrol employed the p53 pathway in regulating stemness, EMT, and metabolic reprogramming. Further investigation of the molecular mechanism of p53 activation by resveratrol may provide useful information for the development of novel therapies for cancer treatment through targeting to CSCs.

Isolation and detection of label-retaining cells in a nasopharyngeal carcinoma cell line

Detection of label retaining cells (LRCs) has been a method to confirm existence of stem cells, and bromodeoxyuridine (BrdU) has commonly been used for labeling. In this study, to verify stem cells in nasopharyngeal carcinoma (NPC), LRCs were established and detected in NPC cell line 5-8F. The 5-8F cells were cultured with BrdU and inoculated subcutaneously into nude mice. By immunohistochemistry, immunocytochemistry, and immunofluorescence, BrdU was detected in 5-8F cells and xenograft tumors. BrdU was strongly positive in cells on the 2nd and the 7th day after being added BrdU, while negative when cells were cultured without BrdU. However, only sporadic cells were positive on the 14th day after BrdU being washed out, and these cells were thought to be LRCs. The average percentage of LRCs was (0.67 +/- 0.32)%. After being cultivated with BrdU for 48 h, 5-8F cells were inoculated into nude mice subcutaneously. After chasing 8 weeks, only sporadic LRCs were detected in xenograft tumors, with a proportion of (0.55 +/- 0.36)%, and these LRCs were located at cancer margin. The existence of LRCs in 5-8F cells indicates the existence of cancer stem cells in NPC.