The Effects Of WNT5A In Metastasis Of Nasopharyngeal Carcinoma And The Effects Of BMI-1 On Chemosensitivity Of Nasopharyngeal Carcinoma Cells

Partâ… WNT5A/PKC Induces Epithelial to MesenchymalTransition and Nasopharyngeal Carcinoma Metastasis[BACKGROUD]Nasopharyngeal carcinoma(NPC) is a prevalent malignancy in South China and Southeast Asia.It is sensitive to radiotherapy and/or radiochemotherapy.The therapeutic effect of NPC patients in early stage is good while it is extremely poor in late stage,and more than 30%of NPCs will develop distant metastases.The underlying mechanism(s) of NPC have been poorly explored.Therefore,it is of great importance to investigate specific moleculars and its mechanism for the guidance of new theraputic targets as well as increasing the 5-year survival rate.WNTs belong to a large family of cysteine-rich secreted glycoproteins consisting of at least 19 members in humans.WNT proteins control cell fate,migration and cellular polarity through cell surface receptors that modulate the transcription of specific target genes. WNT signaling can suppress apoptosis and promote invasive growth of cancer cells.The importance of aberrant WNT signaling has been reported in many human cancers,including NPC.WNT5A signaling has been classified as a noncanonical and nontransforming pathway.There is many evidences that increased WNT5A expression is associated with cancer progression.But little is known about the role and mechanism of WNT5A in the metastasis of NPC.This study aims to explore whether and how WNT5A induces invasion and metastasis of NPC.1) WNT5A is one of the most specific gene in NPC metastasisAmong the cellular clones isolated from CNE-2,Clone-18 has the highest metastatic ability;Clone-22,Clone-26,and the parental CNE-2 line possess limited metastatic potential.Through whole-genome expression profiling of the cultured cells in vitro,we identified 25 up-regulated and 24 down-regulated genes in the high-metastasis Clone-18.Using the same methodology,parallel gene sets in Clone-18 were also identified from xenograft tumors.WNT5A was found to be up-regulated in Clone-18 in both the in vitro and in vivo scenarios.2) Characteristics of epithelial-to-mesenehymal transition(EMT) in Clone-18 cellsMorphologically,the parental line CNE2,as well as Clone-22 and Clone-26,displayed a tightly packed cobblestone appearance.Clone-18 cells,however,displayed a fibroblast-like morphology,suggesting an epithelial-to-mesenchymal transition(EMT).In the Clone-18 cells,the expression of Snail was up-regulated and E-cadherin was down-regulated relative to their expressions in CNE2,Clone-22,and Clone-26 cells, confirming the existence of EMT in the highly metastatic Clone-18 cells.3) Inhibition of Wn5a expression reduced the movement and invasive ability of Clone-18 cellsTo elucidate the role of WNT5A in the motility of NPC cells, three different siRNA sequences targeting WNT5A were designed and tested,siRNA-3 was the most efficient sequence and was chosen for subsequent experiments.Knocking down WNT5A using siRNA-3 dramatically impaired the process of wound healing in Clone-18 cells. Moreover,suppression of WNT5A could significantly reduce the invasive ability of Clone-18 cells(P＜0.05).These results indicate that WNT5A plays a key role in NPC cell migration and invasion.4) Inhibition of PKC activity decreased the motility of Clone-18 cellsThe role of WNT5A in the invasiveness of melanoma cells depends on the Wnt/Ca²⁺ pathway mediated by PKC.It has been reported that PKC activation can up-regulate WNT5A expression in a positive feedback loop in breast cancer cells.We therefore tested whether inhibition of PKC would impede the motility of Clone-18 cells. GF-10923X,a widely used PKC inhibitor,efficiently inhibited the phosphorylation of PKC in Clone-18 cells,producing an inactivated form of PKC.The inhibition of PKC activity by GF-10923X reduced the expression of WNT5A in the Clone-18 cells.The same concentration of GF-10923X also down-regulated the expression of Snail,which is the most prominent EMT-inducing regulator and E-cadherin-specific transcriptional repressor,resulting in the up-regulation of E-cadherin. Consequently,the motility of Clone-18 cells was reduced after GF-10923X treatment.These results were consistently repeated using another conventional PKC inhibitor,G(o|¨) 6983.Therefore,PKC is involved in the EMT of NPC cells,regulating the motility of the cells.5) WNTSA regulates EMT through PKC activityGiven that WNT5A is able to increase the phosphorylation of PKC and that PKC is involved in cell motility and in EMT,we tested whether WNT5A could directly induce EMT.First,we found that knocking down WNT5A mRNA using siRNA-3 in the highly metastatic Clone-18 cells significantly diminished PKC phosphorylation,and also EMT,as indicated by down-regulation of Snail protein and accumulation of E-cadherin in the cells.Second,treatment with recombinant WNT5A protein in the low-metastatic Clone-22 cells resulted in the overexpression of Snail and the reduction of E-cadherin protein.6) Activation of PKC induces motility in low-metastasis cells through Snail and E-cadherin expressionTo determine if activation of PKC can increase cellular motility in the low-metastasis Clone-22 and Clone-26 cells,which possess low levels of WNT5A protein,the clones were treated with the PKC activator PMA.The level of phospho-PKC peaked 30 min after treatment with a low concentration of PMA(200 nM),then gradually decreased to very low levels by 12-24h.Interestingly,both WNT5A and Snail protein levels gradually increased and reached their peaks at 12-24 h,whereas E-cadherin gradually decreased during this period.As expected,cellular motility increased in Clone-22 and Clone-26 cells after treatment with PMA or recombinant WNT5A for 12 h as assessed by the wound healing assay.More importantly,the increase of cellular motility induced by recombinant WNT5A could be abolished by pretreatment with the PKC inhibitor G(o|¨) 6983.7) Up-regulation of WNT5A is associated with NPC metastases in clinical scenariosFinally,we sought to test if the critical role of WNT5A in the migration and invasion of NPC cells revealed in the present study is clinically relevant.Quantitative real-time PCR was performed to evaluate the level of WNT5A mRNA in the tissues of 20 noncancerous nasopharyngeal mucosa,19 primary NPCs,5 metastatic NPC in the cervical lymph nodes(regional metastasis),and 4 metastatic NPC in the liver(distant metastasis).The expression level(relative to GAPDH) of WNT5A mRNA in lymph node metastatic NPC was significantly higher than that in the primary tumor.Further,the expression was much higher in the distant(liver) metastatic NPCs than in regional metastases.There was no significant difference in WNT5A mRNA levels between the noncancerous NP mucosa and primary NPC tissues. [CONCLUSION]1) WNT5A activates the PKC pathway,regulating the expressions of Snail and E-cadherin and subsequently inducing EMT and metastasis in NPC;2) Changes in PKC activity caused by PMA,GF-10923X,or G(o|¨) 6983 can modulate the expressions of Snail and E-cadherin and subsequently affect EMT and metastasis in NPC;3) A positive feedback loop between WNTSA and phosphorylated PKC exists in NPC metastasis; Partâ…¡the Effects of BMI-1 on Chemosensitivity ofNasopharyngeal Carcinoma Cells[BACKGROUD]Nasopharyngeal carcinoma(NPC) is a common malignancy in southern China with uncertain etiologic factors.The development and progression of NPC are believed to result from the interplay of several factors,including genetic susceptibility,Epstein-Barr virus(EBV) infection,and other environmental factors.Currently,the administration of 5-fluorouracil(5-FU)—the most widely used anticancer agent—is one of the standard chemoradio-therapy regimens for NPC.However,a sizable proportion of NPC patients show tumor recurrence after 5-FU treatment that is mainly caused by drug-resistant cancer cells.Therefore, during the past 2 decades,new strategies for enhancing the sensitivity of cancer cells to drug-induced apoptosis for cancer therapy have been intensively explored.The B-cell-specific moloney leukemia virus insert site 1 is one of the components of Polycomb Group protein repression complex 1 (PRC1).It was originally isolated as an oncogene cooperatingwith c-Myc in lymphomagenesis in a murine model.BMI-1 is closely associated with the development and progression of malignant tumors.It is upregulated in a number of cancers and the expression correlates with the invasion and metastasis phenotype,as well as the prognosis of patients.BMI-1 plays critical roles in immortalization of normal epithelial cells and early transformation of malignants,as well as maintenance of the self-renewal of stem cell.Forthermore,some reports showed that BMI-1 is upregulated in nasopharyngeal carcinoma and its expression level is strongly associated with the invasion phenotype of NPC and poor survival of patients.Recently,it has been reported that the downregulation of BMI-1 can result in the apoptosis of cancer cells. Therefore,we hypothesize that the abrogation of BMI-1 expression may be an effective strategy for sensitizing human cancer cells,including NPC cells,to cancer chemotherapy.1) BMI-1 knock down made the ceils more sensitive to 5-FUTo examine the effect of 5-FU on the survival of BMI-1 knock-down cells,an MTT assay was performed after the CNE2-BMI-1/ RNAi,HONE1-BMI-1/RNAi,CNE2-vector,and HONE1-vector cells were treated with 5-FU for 72h.After treatment with various concentrations of 5-FU,we found that both the BMI-1 shRNA-transfected cells,i.e.,the CNE2-BMI-1/RNAi and HONE1-BMI-1/ RNAi cells,showed lower cell viabilities than the empty vector-transfected cells.The IC50 of 5-FU in the CNE2-vector and CNE2-BMI-1/RNAi cells were 9.1504±0.6997 mg/L and 3.1151±0.8073 mg/L(P＜0.05),respectively;the IC50 values of 5-FU in the HONE1-vector and HONE1-BMI-1/RNAi cells were 3.9023±0.752 mg/L and 1.5815±0.433 mg/L(P＜0.05),respectively.These results indicated that BMI-1 knockdown made the cells more sensitive to 5-FU.2) Depletion of BMI-1 enhanced 5-FU-induced apoptosisIn order to evaluate the effect of BMI-1 knockdown on the induction of apoptosis,the CNE2-BMI-1/RNAi and CNE2-vector cells that were treated with 5 mg/mL of 5-FU for 72h were subjected to Hoechst 33258 staining.The cells were examined under a fluorescence microscope.Typical apoptotic morphological changes,such as condensed chromatin,shrunken nuclei,and loss of cell volume,were frequently observed in the CNE2-BMI-1/RNAi cells.Incontrast,only few apoptotic CNE2-vector cells were observed with the same apoptotic morphological changes.In order to further confirm the aforementioned results,we examined the apoptotic rate in the CNE2-BMI-1/RNAi and CNE2-vector cells after treating them with 5 mg/L of 5-FU for 72h.The flow cytometry data showed that the apoptotic rate among the CNE2-BMI-1/RNAi cells was 45.4%as compared to 32.1%among the CNE2-vector cells(P＜0.05).3) PI3K/AKT pathway was essential for the sensitization effect of BMI-1 to 5-FU treatmentTo further explore the mechanism underlying the enhancement of 5-FU-induced apoptosis by the silencing of BMI-1,we examined the expression levels of total-AKT,phospho-AKT,BAX,and BCL-2 in the CNE2-BMI-1/RNAi and CNE2-vector ceils.Our results showed that the knockdown of endogenous BMI-1 led to substantial reduction in the levels of phospho-AKT,while the total-AKT levels remained uninfluenced.Consistent with this reduction in the phospho-AKT level, Westernblot analysis showed significantly decreased expression of BCL-2 in BMI-1-knocked down cells exposed to 5-FU.The accumulation of BAX in the BMI-1-knocked down ceils was more prominent after the cells were exposed to 5-FU.To investigate whether the depletion of BMI-1 enhances 5-FU-induced apoptosis through the PI3K/AKT pathway,the CNE2-BMI-1/RNAi cells were treated with a PI3K inhibitor.The cells were pretreated with 1μM of the PI3K inhibitor wortmarmin for 1 h and then treated with various concentrations of 5-FU; this was followed by the measurement of cell viability determined by the MTT assay.The inhibition rates of the CNE2-BMI-1/RNAi and CNE2-vector cells by 5-FU treatment were not significantly different after application of the PI3K inhibitor.However,the rate of inhibition of the CNE2-vector cells by 5-FU increased after pretreatment of the cells with wortmannin;this was in contrast to the low rate of inhibition of the CNE2-vector cells by 5-FU without influencing PI3K activity.Taken together,the abrogation of the PI3K/AKT pathway could not further increase the sensitivity of the CNE2-BMI-1/RNAi cells to 5-FU treatment.This suggested that PI3K/AKT pathway was essential for the sensitization effect of BMI-1 to 5-FU treatment.[CONCLUSION]1) Knockdown of BMI-1 makes cancer cells more sensitive to 5-FU as well as enhances 5-FU-induced apoptosis.2) Knockdown of BMI-1 inhibits AKT activation and upregulates the expression level of BAX as well as downregulats expression of BCL-23) Knockdown of BMI-1 enhances apoptosis of NPC cells via AKT/PI3K pathway.