The Molecular Mechanism Of JWA As A Functional Molecule To Regulate Cancer Cells Migration

Cell migration is a process that is critical at many stages duringembryonic development, and essential for wound healing and tumor cellinvasion. Cell migration is regulated by coordinated changes in the actincytoskeleton and cell adhesion. Nevertheless, the molecular mechanismsparticipating in the regulation of cell migration remain largely unknown.JWA (AF070523) is a newly identified novel microtubule-associatedprotein (MAP) was essential for the rearrangement of F-actincytoskeleton and activation of MAPK cascades. To elucidate how cellmigration is regulated, we investigated the role of JWA in the migrationand invasion of human tumor cells.Objectives To determine the influence of JWA expression on cellmotility and whether this affect was dependent upon the ability toremodel actin cytoskeleton and activate the MEK-ERK signalingpathway.Methods Henrietta Lacks strain of cancer cells HeLa (human carcinomacervicis cell), B16 (melanoma cell) and HCCLM3 (human hepatoma carcinoma cell) were used for the models of migration and invasion invitro. The intracellular localization of JWA and F-actin was measured byimmunofluorescence microscopy. Tumor cell motility and invasion werenot only evaluated by means of wound-healing migration assay, butassayed by transwell. The involvement of expressions and theirphosphorylations of Raf, MEK and ERK were investigated duringPMA/As₂O₃-induced cell migration. Western blot was used to detect theprotein expression of JWA, FAK and COX-2. Antisense oligonucleotidewas applied to inhibit the expression of JWA and further explore thefunction of it. In order to determine the phosphorylations of MEK/ERKinduced by treatment of As₂O₃ and PMA are dependent on the status ofJWA phosphorylation at SDR or SLR sites, JWA protein PKCphosphorylation site mutation cell strains (ADR-SLR; SDR-ALR;ADR-ALR) were then constructed.Results(1) Our previous studies showed that JWA is a novel microtubulebinding protein and affects microtubule assembly. This implies that JWAmay affect cytoskeleton rearrangement. Since the microtubule associatedproteins (MAPs) were shown to substitute for actin binding proteins inperipheral actin assemblies and induce acting gelatin in vitro, we firsttested hypothesis that JWA may exert an effect on actin assembly. Cellswere transfected with either pEGFP-C1 vector, pEGFP-C1-JWA (sense),or pEGFP-C1-AsJWA (anti-sense). After selection with G418, theresulting HeLa cells were verified and found to possess stable knockdownof JWA expression (knock-down HeLa cells, As-HeLa), stableover-expression of JWA (over-expression HeLa cells, HeLa-JWA) andvector control HeLa (HeLa-vector), respectively. We found that actinfilaments rearrangement was occurred followed by knockdown of JWA alone, the more protrusions were found at the leading edge of theAs-HeLa cells than vector control cells.(2) Both PMA and As₂O₃ were known to as effective model chemicalsto enhance or inhibit cell motility via the activation of MAPK signalcascades and the rearrangement of F-actin cytoskeleton, respectively. Inthis study, cell culture models were established to observe whether JWAaffects PMA or As₂O₃ induced cellular F-actin rearrangement. The resultsshowed that vector only transfected HeLa cells stretched out evenly, andno protrusions were found at the leading edge of the cells after incubationwith PMA for 6 h. The actin filaments were well organized, indicatingthat the HeLa vector cells do not undergo a significant rearrangement ofactin filaments. Upon stimulation with PMA for 12 h, however, thecellular morphology was changed, and the actin filaments wereremodeled. In As-HeLa cells, there were some actin protrusion structuresat the leading edge of the cells upon PMA stimulation within 6 h,indicating that the actin filaments were reorganized. After the stimulationwith PMA for 12 h, the actin filaments underwent a more profoundreorganization. Compared to the vector controls, actin reorganization wasobviously accelerated in these JWA deficient As-HeLa cells. In this study,As₂O₃-induced rearrangements of the actin cytoskeleton were similar tothose induced by PMA. The data above suggest that knock down JWAalone contributes to induce rearrangement of actin filaments, and alsoenhances PMA and As₂O₃ induced actin rearrangement in HeLa cells.(3) Rearrangement of actin filament was usually linked with changedcell motility. To characterize the potential effect of JWA on cell motility,we first examined whether JWA affects the migration of HeLa cells. Theselected cell populations were employed to test for their migrationabilities. Data shown that knockdown of JWA alone significantlypromoted cellular migration when compared with cells carrying control vector. In contrast, over-expression of JWA inhibited cell migration. Thereduced directional persistence suggested that JWA rendered cells unableto maintain a stable direction during targeted migration. Therefore, theseHeLa derivatives were assessed for wound healing migration. Aspredicted, over-expression of JWA produced cells with a marked delay inwound closure, whereas, JWA deficient cells showed accelerated woundclosure. The result from transwell assay was also supported that JWAover expressed HeLa cells reduced the ability to path through the filter,whereas JWA knock-down HeLa cells significantly enhanced themobility. To confirm the biological function of JWA on cell motility, wefurther designed transwell assays in another two kinds cancer cell linesHCCLM3 and B16 cells. As a result, similar phenomena were found,suggesting that the role of JWA on cell migration might exist in multiplecancer cell lines.(4) Since As₂O₃ is also a well-established as anti-proliferative orpro-apoptotic metal, investigations were thus undertaken to determine ifthe anti cell-migration effects of As₂O₃ were due to its anti-proliferationroles. The effect of As₂O₃ on cell cytotoxicity was determined. Theresults showed that significant cytotoxic effects were observed in theserum-supplemented media following the As₂O₃ treatment for 48 h.Exposure of HeLa cells to 2μM As₂O₃ induced approximately 50%growth inhibition. However, in serum-free culture condition, As₂O₃ didnot inhibit cellular proliferation or induce cell apoptosis whenconcentrations under 2μM. However, As₂O₃ was found to exertpronounced cytotoxic effects on HeLa cells in the presence of serum; Thedata in this study was also showed that knock-down JWA was not affectcell viability in HeLa cells after exposed to As₂O₃ within indicatedconcentrations for 48 h when compared to the vector control. To excludethe role of relative inhibition of cell migration in cytotoxicity, the influence of lower concentration As₂O₃ (≤1μM) with serum-free cultureconditions were examined in this study.(5) As₂O₃ treatment alone showed a significant inhibitory effect onHeLa cell migration and in a concentration-dependent manner. In order toevaluate the effects of As₂O₃ in response to PMA accelerated cellmigration behaviors, confluent monolayer of HeLa cells were scrapedwith a pipette tip to remove a section of the monolayer, and the cells werecultured for 24 h with PMA (80 nM) in the presence or absence of As₂O₃.The cell migration rates were assessed by counting the number of cellsthat migrated into the pre-defined denuded area of the cell monolayer.The results showed that As₂O₃ concentration-dependently inhibitedPMA-stimulated HeLa cells migration. Transwell assays also showedsimilar results, in which the ability of the cells to pass through the filterswas measured.(6) The role of JWA on chemicals induced migration of HeLa cellswere also determined by using transwell assays. Data showed thattreatment of the cells with As₂O₃ markedly inhibited PMA-stimulated cellmigration and in a concentration-dependent manner. However, thisinhibitory effect of As₂O₃ was significantly blocked in JWA deficientHeLa cells. In contrast, JWA deficiency further enhancedPMA-stimulated cell migration. Suggesting that intracellular expressionof JWA is important for the effects of As₂O₃ on PMA induced cellmigration.(7) It is well known that both As₂O₃ and PMA treatment inducedactivation of MAPK cascade. JWA was also proposed as a putativeMAPK activated protein. Therefore, it was hypothesized that JWA mightaffect cell migration via MAPK cascade. To test this hypothesis, weconducted western blot analysis for MEK-ERK expressions and theirphosphorylation status in cell migration models. The effects of JWA transfection on RAF-MEK-ERK phosphorylation in HeLa cells were firstinvestigated. Data showed that, without PMA or As₂O₃ treatment, thecells with vector control, knockdown JWA expression or followed rescuetransfection were no significant phosphorylation can be detected for thesesignal molecules of MAPK cascades. The phosphorylations ofRaf-MEK-ERK occurred in vector only transfected cells after treatmentwith either PMA or As₂O₃ or the both, however, the phosphorylations ofMEK-ERK but not Raf were completely blocked in JWA deficient HeLacells. These data provide evidence that knockdown of JWA enhanced cellmigration might be mediated by MEK-ERK associated signal pathway.(8) To further confirm whether JWA was essential for thephosphorylation of MEK-ERK induced by As₂O₃/PMA, we designedJWA rescue cell culture models. JWA-deficient HeLa cells weretransiently transfected with pEGFP-C1-JWA to produce JWA-rescuedHeLa cells which recovered JWA expression. In the serum-free cellmigration models, JWA-rescued HeLa cells and vector-control HeLacells were treated with PMA (80 nM) for 12 h in the presence or absenceof As₂O₃ (1μM) for a further 12 h, and subsequently the phosphorylationstatus of Raf-MEK-ERK was determined. We found that As₂O₃ or PMAtreatment triggered MEK and ERK phosphorylations were completelyrecovered in the cells presence of rescued JWA expression. Interestingly,JWA rescue did not influence the activation of Raf.(9) Both As₂O₃ and PMA induced similar activation patterns of theMEK-ERK cascade and played an essential role in cell migration. Basedon these experimental facts, the questions arose as to how similarMEK-ERK phosphorylation patterns mediate reversal effects on cellmigration? A reasonable explanation might be that As₂O₃ and PMAactivating different downstream substrates of ERK, separately, whichmay involve different phosphorylation sites of these MAPK cascades molecules. To test our hypothesis, we selected COX-2 and FAK as thedifferential downstream target responsive to PMA and As₂O₃,respectively, and determined their expressions in As₂O₃ or PMA-inducedHeLa cell migration model. Data showed that, after PMA (80 nM)stimulation, the expression of COX-2 was markedly up-regulated in JWAknockdown HeLa cells accompanied by no changes in vector-controlcells and no influence on FAK. In contrast, As₂O₃ (1μM) treatment onlyled to up-regulation of FAK not COX-2 expressions. These data suggestthat JWA plays a key role in PMA/As₂O₃ via MEK-ERK-dependantpathway which regulates COX-2/FAK separately and linked to thecontrasting effects on cell migration.(10) To further elucidate the role of JWA in these consequent events,we employed JWA rescue cell culture models. Treatment ofJWA-rescued HeLa cells and HeLa-vector cells with PMA (80 nM) for12 h was conducted in the presence or absence of As₂O₃ (1μM) forfurther 12 h; subsequently, the expressions of COX-2 and FAK weredetermined. We found that recovered JWA expression in JWA-deficientHeLa cells significantly reversed expression patterns of COX-2 by PMAand FAK by As₂O₃ treatments. These results supported the postulationthat JWA plays a critical role in activation of the MEK-ERK downstreamas a consequence of As₂O₃ and PMA stimulation.(11) In order to determine whether JWA serves as a kinase-likemolecule in MAPK cascade, we constructed series JWA expressionvectors with PKA/PKC phosphorylation sites mutants which were thentransiently transfected into JWA knockdown HeLa cell lines, respectively.Therefore, three JWA protein PKC phosphorylation site mutation cellstrains (ADR-SLR; SDR-ALR; ADR-ALR) were then constructed,expressed as As-HeLa-P1, As-HeLa-P2 and As-HeLa-P12, respectively.These conditional cell lines were used for determine if the phosphorylations of MEK/ERK induced by treatment of As₂O₃ and PMAare dependent on the status of JWA phosphorylation at SDR or SLR sites.JWA vector control and phosphorylation site-mutated HeLa cells weretreated with PMA (80 nM) for 12 h in the presence or absence of As₂O₃(1μM) for further 12 h, the phosphorylations of MEK or ERK and itsdownstream effectors FAK and COX-2 were determined. Data showedthat, in As-HeLa-P1 HeLa cells, As₂O₃ and PMA treatments were stillable to induce activation of MEK and ERK. Further, the expression ofCOX-2 by PMA and FAK by As₂O₃ was almost rescued. However,As-HeLa-P2 cells showed a reduced level of activation of MEK and ERK.It is of interest that, in As-HeLa-P12 cells, As₂O₃ or PMA treatment wascompletely unable to induce activation of MEK and ERK. Moreover,PMA-induced up-regulation of COX-2 and As₂O₃-induced down-regulation of FAK was also not reversible in As-HeLa-P12 cells.However, JWA phosphorylation site mutation did not influence theactivation of Raf. Taken together, these studies indicate that thephosphorylation motif of JWA is critical for activation of the MEK-ERKpathway induced by either As₂O₃ or PMA.(12) To further investigate the role of JWA phosphorylation on cellmigration, As-HeLa cells were transfected with a series pEGFP-N1-JWAPKC phosphorylation sites mutation vectors, or pEGFP-C1-JWA,separately. After selection with G418, pools of cells that phosphorylationsite mutation JWA (As-HeLa-P12) and JWA rescue proteins (As-HeLa-rescue) were generated. Transwell assays showed that in As-HeLa-rescued cells, the facilitative effect of JWA deficiency on cell migrationby PMA and As₂O₃ was significantly rescued. Interestingly, the cells withJWA mutants (As-HeLa-P12) inversely stimulated the migration either byAs₂O₃ or PMA. Therefore, the existence of SDR-SLR motifs in JWAprotein is critical for regulating cell migration. Conclusion JWA, a newly identified novel microtubule-associatedprotein (MAP) was essential for the rearrangement of F-actincytoskeleton and activation of MAPK cascades induced by arsenictrioxide (As₂O₃) and phorbol ester (PMA).All the data suggest that JWA inhibits random and directed tumor cellmigration. JWA may initiate the regulatory pathways, such as regulationof MT and actin cytoskeleton, leading to intensive migration.In HeLa cells, knock down JWA alone contributes to inducerearrangement of actin filaments, and also enhances PMA and As₂O₃induced actin rearrangement. JWA plays a critical role in activation of theMEK-ERK downstream as a consequence of As₂O₃ and PMA stimulationand links to the contrasting effects on cell migration. JWA may befunctional as a kinase-like molecule in MAPK cascade; the SDR-SLRphosphorylation motifs play a critical role in MAPK pathway mediatedtumor cells migration.Taken together, JWA, works as a potential critical molecule, associatedwith migration and invasion of human tumor cells, and may be a novelmolecular marker for it. A full understanding of JWA function mayprovide significant insights into the pathogenesis, development andprognostic of tumor.