Epstein-Barr Virus-encoded LMP2A Stimulates Migration Of Nasopharyngeal Carcinoma Cells Via EGFR/Ca²⁺/Calpain/ITG?4 Axis

Nasopharyngeal Carcinoma(NPC)is a malignant tumor that occurs in the nasopharyngeal mucosa.It is generally believed that the occurrence and development of NPC is a multi-path and multi-step process of interaction between genomes-environment and Epstein-Barr virus(EBV)infection.In the high incidence of NPC,almost 100% of the NPC patients are EBV positive.EBV genome is expressed in all NPC cells of the patient,mainly type II latent state,expressing three latent membrane proteins(LMP1,LMP2 A,LMP2B),EBV nuclear antigen 1(EBNA1)and EBV encoded small RNAs(EBERs).These viral products contribute to the promotion of epithelial-mesenchymal transition(EMT)of NPC cells,leading the tumor to a greater metastatic potential.At the transcriptional level,the detection rate of LMP2 A in NPC was as high as 90%.Our previous study found that LMP2 A expression of NPC cellsenhance the ability to migrate and invasion,and induce localization and aggregation changes of integrin ITG?4.And the change localization of ITG?4may lead to the collapse of the hemidesmosomes,which contributes to the migration of cells.This study explored and confirmed the molecular mechanism of LMP2 A expression inducing changes in ITG?4 localization in order to elucidate the molecular mechanism of LMP2 A promoting the metastasis of NPC from a new perspective.First of all,we successfully constructed NPC cell lines CNE1 and TW03 that stably expressed LMP2 A.The fluorescent Ca2+ indicator was used to detect the relative amount of intracellular Ca2+,we found that when expressing LMP2 A,CNE1 and TW03 have higher content of Ca2+,and Ca2+ content was significantly increased under the action of EGF.In order to investigate the role of intracellular Ca2+ in cell migration,we used Ca2+ chelator BAPTA-AM to block intracellular free Ca2+ and found that the migration ability of LMP2A-CNE1 and LMP2A-TW03 was significantly attenuated,suggesting that LMP2 A may be dependent on the increase of intracellular Ca2+ thereby promoting migration of NPC cells.Secondly,in order to investigate the influence of LMP2 A to intracellularCa2+.We analyzed the relationship between LMP2 A expression and EGFR expression and its phosphorylation.We found that the phosphorylation of EGFR in LMP2A-CNE1 and LMP2A-TW03 was higher than that in control cells.Immunofluorescence staining was used to detect the positionchanged of EGFR.These results indicate that the expression of LMP2 A in NPC cells activates the EGFR pathway.Thirdly,in order to investigate the downstream molecular pathways that LMP2 A affects cell metastasis through regulation of Ca2+,we examined the theactivity of the Ca2+-dependent protease,calpain(CAPNs),by calpain activity assay kit.In concordance with cytosolic Ca2+,we observed a higher calpain activity in LMP2A-CNE1 and LMP2A-TW03 cells,and the difference was statistically significant.This suggests that expression of LMP2 A in NPC cells activates EGFR,increases intracellular calcium and thus activates calpain.In the interest of elucidating the effect of calpain activity on the migration of NPC cells,we employed a calpain inhibitor SJA6017 in a wound healing assay.The results showed that the SJA6017 significantly reduced the motility of these LMP2 A positive NPC cells,in a dose-dependent manner.This suggests that the expression of LMP2 A promotes NPC cell migration via EGFR/Ca2+/calpain axis.Finally,in order to elucidate the association of CAPNs mediated cleavage of ITG?4 with cell mobility,we analyzed the localization of ITG?4 in TW03 and LMP2A-TW03 by laser confocal microscopy.It was found that ITG?4 in TW03 cells was evenly distributed at the basal layer of cells,while aggregated at the cellular filopodia in LMP2 A positive TW03 cells.In order to verify that the change of ITG?4 in LMP2A-TW03 cells was closely related to the activity of calpain,we further treated LMP2A-TW03 cells with SJA6017,and the expression of ITG?4 was restored to the state of cell bottom,similar to TW03 cells.In addition,we also found that ITG?4(205kD)can be cleaved into two subtypes(165k Da and 125kDa)by Western blot.In the positive expression of LMP2 A CNE1 and TW03,the cleavage of ITG?4 was significantly higher than the control cell line,and SJA6017 can inhibit the occurrence of ITG?4 cleavage.All these suggest that protein kinase-dependent cleavage of ITG?4 may be related to its changes of localization and thus promote cell migration.In summary,EBV-encoded LMP2 A may promote the metastasis of NPCcells by activating EGFR,increasing calcium content,activating CAPNs activity,thus the cleavage of ITG?4 and promote change of ITG?4 membrane location.Thus,we found a new molecular mechanism to promote the migration of NPC cells.Our data shed light on how the EBV encoded LMP2 A protein may contribute to the metastasis properties of NPC tumor cells and point to possible drug targets for controlling NPC cell migration,has far-reaching significance for effective prevention and treatment of nasopharyngeal carcinoma.