Invasion Of Nasopharyngeal Cancer Cells Research Articles

Tumor cell-derived exosomal miR-193b-3p promotes tumor-associated macrophage activation to facilitate nasopharyngeal cancer cell invasion and radioresistances

BackgroundCommunication between cancer cells and tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) plays a crucial role in accelerating nasopharyngeal cancer (NPC) metastasis and radioresistance. However, the mechanisms through which NPC cells regulate the properties and activation of TAMs during NPC progression are not yet fully understood. MethodsA high-metastatic NPC subclone (HMC) and a low-metastatic NPC subclone (LMC) were screened from the CNE-2 cell line and exosomes were collected from HMCs and LMCs, respectively. The effects of HMC- and LMC-derived exosomes (HMC-Exos and LMC-Exos) on the regulation of TAM activation were evaluated by assessing the levels of inflammation-related or immunosuppression-related genes. The role of miRNA-193b-3p (miR-193b) in mediating communication between NPCs and TAMs was assessed using real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot analysis, Transwell assays, and clonogenic survival assays. ResultsHMCs and HMC-Exos exhibited a greater capacity to facilitate macrophage protumorigenic activation than LMCs and LMC-Exos. miR-193b levels derived from HMC-Exos were higher than those from LMC-Exos, and miR-193b levels were higher in metastatic NPC tissue-derived TAMs than in non-metastatic NPC tissue-derived TAMs. The upregulated miR-193b was packaged into exosomes and transferred to macrophages. Functionally, miR-193b up-regulation accelerated TAM activation by directly targeting mitogen-activated protein/ERK kinase kinase 3 (MEKK3). As a result, miR-193b-overexpressed macrophages facilitated NPC cell invasion and radioresistance. ConclusionsThese data revealed a critical role for exosomal miR-193b in mediating intercellular communication between NPC cells and macrophages, providing a potential target for NPC treatment.

KIF15, a key regulator of nasopharyngeal carcinoma development mediated by the P53 pathway

Background: Kinesin family member 15 (KIF15) is a protein that regulates cell mitosis and plays an important role in the development and progression of several types of human cancers. However, the role of KIF15 in the development of nasopharyngeal cancer (NPC) is still unclear. Methods: The differential expression of KIF15 in NPC and para-carcinoma tissues was evaluated based on data collected from Gene Expression Omnibus (GEO) database and immunohistochemical analysis of clinical specimens collected from a patient cohort. Cell lines 5-8F and CNE-2Z were selected for the construction of KIF15‑knockdown cell models. CCK8 assay, flow cytometry, wound healing, Transwell and clone formation assays were used to detect the proliferation, apoptosis, migration, invasion and colony formation of NPC cells in vitro. A mouse xenograft model and the tail intravenous mouse distant transfer model were constructed for in vivo study. Furthermore, the potential molecular mechanisms underlying the effects of KIF15 were explored through western blot analysis, and several in vitro and in vivo functional assays were performed to explore its role in NPC. Results: The results revealed significantly higher expression of KIF15 in NPC tissues compared to para-carcinoma tissues. High levels of KIF15 expression were also associated with short overall survival (OS) and progression-free survival (PFS). Knockdown of the KIF15 gene led to a cell cycle arrest in the growth 2 (G2) phase, inhibition of cell proliferation, migration, invasion, colony formation, and enhanced cell apoptosis. The in vivo murine xenograft experiments showed that down-regulation of the KIF15 gene could inhibit tumor growth and reduce the risk of liver and lung metastasis in NPC. Moreover, the evaluation of the molecular pathway showed that the mitogen-activated protein kinase/P53 pathways might be involved in the KIF15-induced regulation of NPC. Rescue assays indicated that Pifithrin-α could counteract the pro-proliferative and pro-apoptotic effects mediated by KIF15. Conclusion: This work indicated that KIF15 overexpression accelerated the progression of NPC and promoted the development of distant metastases. Therefore, KIF15 may have an important role as a prognostic indicator and a potential drug target for the treatment of NPC.

Proliferation, Metastasis, and Radiosensitivity of Nasopharyngeal Carcinoma Cells in the Expression and Effect of Kiwifruit Extract through the Regulation of miR-205-5p.

Background Radix Actinidiae extract (RAE) has been shown to inhibit cancer in many studies, but its potential mechanism in nasopharyngeal cancer (NPC) progression remains unclear. Methods NPC cells (SUNE1) were treated with different doses of RAE. For transfection, SUNE1 cells were transfected with the microRNA (miR)-205-5p inhibitor (anti-miR-205-5p) or mimic followed by treatment with 200 μg/mL RAE for 24 h. The MTT assay and colony formation assay were used to detect cell proliferation and radiosensitivity. The transwell assay was used to detect cell migration and invasion. The expression of miR-205-5p was detected by quantitative real-time PCR. The protein expression levels of matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) were detected by western blot analysis. Results RAE inhibited NPC cell proliferation, migration, and invasion, while it enhanced radiosensitivity (P < 0.05). Also, RAE treatment decreased miR-205-5p expression, as well as MMP2 and MMP9 protein levels (P < 0.05). Anti-miR-205-5p transfection enhanced the effects of RAE on NPC cell proliferation, migration, invasion, and radiosensitivity (P < 0.05), while miR-205-5p mimic transfection had an opposite effect (P < 0.05). Conclusion RAE might decrease miR-205-5p, thereby it inhibited NPC cell proliferation and metastasis and enhanced radiosensitivity.

Long non-coding RNA GNAS-AS1 promotes cell migration and invasion via regulating Wnt/β-catenin pathway in nasopharyngeal carcinoma.

The diagnosis and prognosis of nasopharyngeal cancer (NPC) are still difficult. To investigate the effect of long-chain non-coding RNA GNAS-AS1 (lncRNA GNAS-AS1) on proliferation, migration, and invasion of NPC, we carried out this research to illustrate the underlying mechanism. Real-time quantitative PCR was used to detect the expression of GNAS-AS1 in nasopharyngeal carcinoma cells. The effect of transfection of si-GNAS-AS1 on the growth of nasopharyngeal carcinoma SUNE-1 cells was analyzed by CCK-8 assay and colony formation assay. The effect of GNAS-AS1 on the migration and invasion of SUNE-1 cells was detected by transwell assay and Matrigel assay. The expression of C-myc, CyclinD, and MMP2 was detected by Western blot. The expression of β-catenin was detected by real-time quantitative PCR and Western blot. GNAS-AS1 was upregulated in NPC. GNAS-AS1 promoted cell proliferation, cell migration, and cell invasion in vitro. GNAS-AS1 exerted its function by regulating Wnt/β-catenin pathway. GNAS-AS1 functioned as an oncogenic role via mediating β-catenin expression. LncRNA GNAS-AS1 played an important role in the proliferation, migration, and invasion of NPC cells, suggesting that GNAS-AS1 may be an important gene related to the formation and progression of nasopharyngeal carcinoma. The completion of this study provides new potential therapeutic targets for nasopharyngeal carcinoma.

MiRNA-506 presents multiple tumor suppressor activities by targeting EZH2 in nasopharyngeal carcinoma

ObjectiveMiR-506 has been reported to be associated with multiple malignancies, but its roles in nasopharyngeal cancer (NPC) are not fully understood. Our objective is to demonstrate its effects on NPC and the underlying mechanisms. MethodsTotally fifteen pairs of NPC and adjacent non-tumorous tissues were collected for the detection of miR-506 and enhancer of zeste homolog 2 (EZH2) expression. Dual luciferase reporter assay was employed for verifying the relationship between miR-506 and EZH2. The flow cytometry and MTT assays were employed to explore the effects of miR-506 and EZH2 on the cell apoptosis and proliferation, respectively. Wound closure and transwell assays were used to evaluate the cell migration and invasion abilities. Western blotting or RT-qPCR assays were applied to detect the alterations of miR-506, EZH2 and epithelial-mesenchymal transition (EMT)-related markers. Morphological changes of cells with EMT were assessed by light microscopy. ResultsMiR-506 was significantly decreased and EZH2 was obviously increased in NPC tissues. Overexpression of miR-506 decreased the EZH2 level, promoted apoptosis, inhibited proliferation, invasion and migration of NPC cells. Accordingly, miR-506 overexpression attenuated EMT process of NPC cells as demonstrated by the alterations of EMT-related markers and the morphological changes. In addition, the luciferase assay proved that miR-506 directly targeted EZH2. Furthermore, the overexpression of EZH2 reversed the tumor-suppressive effects induced by miR-506 mimics. ConclusionMiR-506 acted as a tumor suppressor to promote apoptosis and inhibit invasion and migration via directly targeting EZH2. MiR-506 can be a candidate target for gene therapy against NPC.

Circular RNA circ_0000615 knockdown suppresses the development of nasopharyngeal cancer through regulating the miR-338-3p/FGF2 axis.

Nasopharyngeal cancer (NPC) is a type of head and neck cancer with a high rate of metastasis. Circular RNAs (circRNAs) were reported to be related to the development of human cancers. This research aimed to investigate the functional mechanism of circRNA circ_0000615 in NPC. The gene expression was examined by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to assess cell proliferation ability. Transwell assay was used to measure cell migratory and invasive abilities. Furthermore, the interaction between miR-338-3p and circ_0000615 or fibroblast growth factor 2 (FGF2) was predicted by starBase v.2.0 and then confirmed by the dual-luciferase reporter assay. Besides, the mouse xenograft experiment was carried out to explore the effect of circ_0000615 on tumor growth in vivo. We detected increased levels of circ_0000615 and FGF2, along with a decreased level of miR-338-3p in NPC tissues and cells. Circ_0000615 knockdown suppressed the proliferation, migration, invasion, and EMT of NPC cells. Interestingly, circ_0000615 interacted with miR-338-3p, and miR-338-3p targeted FGF2. Circ_0000615 inhibited miR-338-3p expression to upregulate the FGF2 level. Furthermore, both miR-338-3p depletion and FGF2 overexpression weakened the effect of circ_0000615 knockdown on NPC cell progression. Besides, circ_0000615 knockdown repressed tumor growth in vivo. In conclusion, our findings demonstrated that circ_0000615 knockdown suppressed the growth of NPC cells via modulating miR-338-3p/FGF2 axis, providing a theoretical basis for the treatment of NPC.

PD-L1 Induces Epithelial‐Mesenchymal Transition in Nasopharyngeal Carcinoma Cells Through Activation of the PI3K/AKT Pathway

Nasopharyngeal cancer (NPC) is a malignant epithelial carcinoma of the head and neck. Cancer therapy targeting programmed cell death protein-1 (PD-1) or programmed death ligand-1 (PD-L1) is revolutionary. However, the tumorigenic mechanism of PD-L1 is not yet clear in NPC. Here we demonstrated an oncogenic role of PD-L1 via activating PI3K/AKT in NPC cells. PD-L1 overexpression was frequently detected in NPC biopsies and cell lines by qRT-PCR. PD-L1 overexpression and knockdown demonstrated that PD-L1 promoted NPC cell invasion and metastasis in vitro and in vivo. Mechanistically, PD-L1 prominently activated the epithelial–mesenchymal transition (EMT) process in a PI3K/AKT-dependent manner. Taken together, we found that PD-L1 overexpression confers NPC cell malignancy and aggressiveness via activating the downstream PI3K/AKT signaling. Thus, these results provide a basis for diagnosis and treatment of NPC.

Folic acid inhibits nasopharyngeal cancer cell proliferation and invasion via activation of FRα/ERK1/2/TSLC1 pathway.

Folic acid (FA), which is necessary for normal cell division of mammals, has been implicated to be involved in many tumors. Dietary FA intake has been reported to be associated with a lower risk of nasopharyngeal cancer (NPC). However, the molecular mechanisms of FA in NPC cells remain unclear. In the present study, we found that FA treatment dose dependently inhibited the proliferation, invasion and migration of NPC cells, via folate receptor α (FRα). We further found that FA, bound to FRα, induced the activation of MEK/ERK1/2, and increased the expressions of TSLC1 and E-cadherin. Moreover, blocking of ERK1/2 activation attenuated FA-mediated increase in TSLC1 expression. In addition, knockdown of TSLC1 abolished the FA-mediated inhibition of cell proliferation, invasion and migration, and suppressed the FA-mediated increase oinE-cadherin expression in NPC cells. Taken together, our data suggest that FA treatment inhibits NPC cell proliferation and invasion via activation of FRα/ERK1/2/ TSLC1 signaling pathway. Therefore, FA could be explored as a therapeutic drug for the treatment of NPC, and TSLC1 may act as a tumor suppressor in NPC.

TBX2 over-expression promotes nasopharyngeal cancer cell proliferation and invasion.

TBX2 is a member of the T box transcription factor family. Its expression and potential biological functions in nasopharyngeal cancer (NPC) cells are studied here. We showed that TBX2 mRNA and protein expression was significantly elevated in multiple human NPC tissues, as compared with that in adjacent normal tissues. Knockdown of TBX2 by targeted-siRNA significantly inhibited proliferation and invasion of NPC cells (CNE-1 and HONE-1 lines). Meanwhile, TBX2 knockdown also induced G1-phase cell cycle arrest. At the molecular level, we discovered that expressions of several tumor suppressor genes, including p21, p27, phosphatase with tensin homology (PTEN) and E-Cadherin, were increased dramatically after TBX2 knockdown in above NPC cells. Collectively, our results imply that TBX2 over-expression promotes NPC cell proliferation and invasion, possibly via silencing several key tumor suppressor genes.

MiR-346 promotes migration and invasion of nasopharyngeal carcinoma cells via targeting BRMS1.

The aim of this study is to determine the expression and roles of miR-346 in nasopharyngeal carcinoma (NPC). We showed that miR-346 was upregulated in NPC tissues compared with adjacent non-tumorous nasopharyngeal tissues. Inhibition of miR-346 significantly attenuated the migration and invasion of NPC cells. Luciferase reporter assay showed that miR-346 targeted the 3'-untranslated region (3'-UTR) of breast cancer metastasis suppressor 1 (BRMS1). Overexpression of miR-346 suppressed the endogenous expression of BRMS1 in NPC cells. There was a significant negative correlation between miR-346 and BRMS1 protein expression in NPC tissues (r=-0.372, P=0.008). Rescue experiments demonstrated that overexpression of BRMS1 lacking the 3'-UTR impaired the invasiveness of NPC cells transfected with miR-346 mimic. Taken together, miR-346 shows the ability to promote the migration and invasion of nasopharyngeal cancer cells via targeting BRMS1 and represents a potential therapeutic target for NPC.

Alteration of the Methylation Status of Urokinase Plasminogen Activator (uPA) is involved in Proliferation and Invasion of Nasopharyngeal Cancer Cells

The extracellular matrix degradation is the most important step in the process of tumor cell metastasis. Urokinase-type plasminogen activator (uPA) can catalyzes the conversion of the inactive zymogen plasminogen to the active broad-spectrum plasmin, which degrades a number of matrix proteins and also activates other proteases, including some matrix metalloproteinases.

Met inactivation by S-allylcysteine suppresses the migration and invasion of nasopharyngeal cancer cells induced by hepatocyte growth factor

PurposePast studies have reported that S-allylcysteine (SAC) inhibits the migration and invasion of cancer cells through the restoration of E-cadherin, the reduction of matrix metalloproteinase (MMP) and Slug protein expression, and inhibition of the production of reactive oxygen species (ROS). Furthermore, evidence is emerging that shows that ROS induced by radiation could increase Met activation. Following on these reports of SAC and Met, we investigated whether SAC could suppress Met activation.Materials and MethodsWound healing, invasion, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT), soft agar colony forming, western blotting, and gelatin zymography assays were performed in the human nasopharyngeal cancer cell lines HNE1 and HONE1 treated with SAC (0, 10, 20, or 40 mM) and hepatocyte growth factor (HGF).ResultsThis study showed that SAC could suppress the migration and invasion of HNE1 and HONE1 cell lines by inhibiting p-Met. An increase of migration and invasion induced by HGF and its decrease in a dose dependent manner by SAC in wound healing and invasion assays was observed. The reduction of p-Met by SAC was positively correlated with p-focal adhesion kinase (p-FAK) and p-extracellular related kinase (p-ERK in both cell lines). SAC reduced Slug, MMP2, and MMP9 involved in migration and invasion with the inhibition of Met-FAK signaling.ConclusionThese results suggest that SAC inhibited not only Met activation but also the downstream FAK, Slug, and MMP expression. Finally, SAC may be a potent anticancer compound for nasopharyngeal cancer treated with radiotherapy.

Inhibition of c-Met downregulates TIGAR expression and reduces NADPH production leading to cell death

c-Met represents an important emerging therapeutic target in cancer. In this study, we demonstrate the mechanism by which c-Met tyrosine kinase inhibition inhibits tumor growth in a highly invasive Asian-prevalent head and neck cancer, nasopharyngeal cancer (NPC). c-Met tyrosine kinase inhibitors (TKIs; AM7 and c-Met TKI tool compound SU11274) downregulated c-Met phosphorylation, resulting in marked inhibition of NPC cell growth and invasion. Strikingly, inhibition of c-Met resulted in significant downregulation of TP53-induced Glycolysis and Apoptosis Regulator (TIGAR) and subsequent depletion of intracellular NADPH. Importantly, overexpression of TIGAR ameliorated the effects of c-Met kinase inhibition, confirming the importance of TIGAR downregulation in the growth inhibitory activity of c-Met TKI. The effects of c-Met inhibition on TIGAR and NADPH levels were observed with two different c-Met TKIs (AM7 and SU11274) and with multiple cell lines. As NADPH provides a crucial reducing power required for cell survival and proliferation, our findings reveal a novel mechanistic action of c-Met TKI, which may represent a key effect of c-Met kinase inhibition. Our data provide the first evidence linking c-Met, TIGAR and NADPH regulation in human cancer cells suggesting that inhibition of a tyrosine kinase/TIGAR/NADPH cascade may have therapeutic applicability in human cancers.

STAT3 activation contributes directly to Epstein‐Barr virus–mediated invasiveness of nasopharyngeal cancer cells in vitro

Nasopharyngeal cancer (NPC) is an Epstein-Barr virus (EBV)-associated head and neck cancer prevalent in Asia. Although with reasons not fully understood, the intrinsic invasiveness of NPC is believed to be EBV-linked. Recently, EBV was found to induce STAT3 activation. Constitutive STAT3 activation correlated with advanced clinical staging in NPC. We hypothesized that STAT3 activation by EBV directly contributes to the intrinsic invasiveness of NPC cells. Phospho-STAT3-Tyr705 was detected in high percentage of NPC tumors (7/10 cases). Using a paired NPC cell line model, HONE-1 and the EBV-infected counterpart, HONE-1-EBV, we found that HONE-1-EBV expressed a higher level of phospho-STAT3-Tyr705 and was approximately 11-fold more invasive than HONE-1. In HONE-1-EBV, STAT3 siRNA targeting inhibited both spontaneous and serum-induced invasion, as well as cell growth. Conversely, activation of STAT3 (by expressing an activated STAT3 mutant, namely STAT3C) in the parental HONE-1, mimicking EBV-induced STAT3 activation, significantly enhanced its invasiveness and proliferation, which was accompanied by increased expression of markers of mesenchymal status, proliferation and anti-apoptosis. Our results demonstrated that EBV-induced STAT3 activation is responsible for NPC cell proliferation and invasion. This was further confirmed by a small molecule inhibitor of JAK/STAT3, JSI-124. JSI-124 inhibited STAT3 activation in HONE-1-EBV, with subsequent growth inhibition, induction of PARP cleavage, abrogation of anchorage-independent growth and invasion. We found that EBV-independent activation of STAT3 by a growth factor, EGF, also contributed to NPC invasion. In conclusion, EBV-induced STAT3 activation directly contributes to the intrinsic invasiveness of NPC cells and STAT3 targeting may be beneficial in treating aggressive NPC.