EGF Enhances The Migration Of Cancer Cells By Up-regulation Of TRPM7

Lung cancer is one of the most malignant cancers. Among differenttypes of lung cancers, non-small cell lung cancer (NSCLC) accounts for75–80%of all the cases. Despite extensive research in diagnostic andtreatment strategies, the5-year survival rate for lung cancer patients is only8–14%. The major reasons for the poor survival rate include the propensity ofearly metastasis and the lack of efficient therapeutic strategy for the metastasisdue to the poor understanding of its mechanism. Thus, an improvedunderstanding of the molecular mechanisms for the metastasis of NSCLD willlead to improved therapeutic approaches and prognosis for patients.Cell migration is required for the invasion and metastasis of tumor cells.The ubiquitous second messenger Ca²⁺plays an important role in cellmigration. However, the roles of Ca²⁺are complicated and have not been fullyelucidated. It is known that cell migration needs the presence of extracellularCa²⁺. Thus, the identities of Ca²⁺pathways in the plasma membrane, which areinvolved in Ca²⁺influx for the cell migration, are naturally a focus of thestudies. In this regard, recent works on transient receptor potential (TRP)channels are interestingly noticeable. TRP channels are non-selective cationchannels and pathways for Ca²⁺entry in various non-excitable and excitablecells. The TRP channel superfamily includes TRPC, TRPM, TRPV and othersubfamilies. Many TRP channels have been found in cancer cells. In cancerA549cells (a human lung adenocarcinoma epithelia cell line), the mRNAexpressions of TRPC1, TRPC3, TRPC6, TRPC7, TRPM7have been reported,with the expression level of TRPM7being the highest. TRPM7is a member ofTRP family with dual-function (ion channel/protein kinases) and predominantpermeability for Ca²⁺and Mg2+. Recent studies show that TRPM7is importantfor platelet-derived growth factor (PDGF) induced proliferation and migration of human osteoblasts and the migration of embryonic lung fibroblast andhuman nasopharyngeal carcinoma5-8F cells. Based on these studies, wehypothesize that TRPM7channel may play an important role in the migrationof A549cells.Epidermal growth factor receptor (EGFR) is highly expressed in somecancer cells, and its activity is believed to be important for the malignantproperties of the cancer cells including the invasion and metastasis. Therecently developed blockers of EGFR, such as Iressa and Erbitux have beensuccessfully used for the treatment of cancers, especially of lung cancers. Itwill be interesting to know whether TRPM7could be the target of EGFRmodulation. Up to now, the functional expression of TRPM7channels in lungcancer cells has not been studied. In this study, we used a combination ofelectrophysiology, Western blot and immunofluorescence techniques toinvestigate the presence and functional significance of TRPM7in A549cells.Our results suggest a functional expression of TRPM7which is important forthe migration of A549cells and is also a target for EGF modulation. Overall,our results indicate that inhibition of TRPM7could be an effective therapeuticapproach for NSCLD.Part1Functional expression of TRPM7in lung cancer cellsObjective: To investigate the functional expression of TRPM7in lungcancer cell.Methods: RT-PCR,Western blot,immunohistochemistry, immunocyt-chemistry and confocal microscopy technique were used to investigate themRNA and protein expression of TRPM7in A549cells and in the tissues ofhuman lung adenocarcinoma. Whole-cell patch-clamp technique was used tomonitor the TRPM7-like current. TRPM7inhibitor2-APB andTRPM7-shRNA were used to investigate the molecular identities of theTRPM7-like current.Results:1Characterization of TRPM7-like currents in A549cellsWe first used the whole-cell patch-clamp technique to investigate the endogenous whole-cell current characteristics in A549cells. The currentswere readily elicited in A549cells by a ramp voltage protocol ranging from120to+120mV after dialysis of cells by Mg2+-free pipette solution. Thecurrents showed characteristics of strong outward rectification in the presenceof extracellular Ca²⁺, and with little inward currents seen at negativemembrane potentials. The outward currents developed slowly and reached asteady state in about10min after the patch rupture. The current–voltagerelationship with small inward currents and strong outward rectification issimilar to that of recombinant TRPM7currents. A substantial component ofinward currents was activated by removing Ca²⁺from the extracellularsolution. The current density of TRPM7-like current in extracellular solutionwith1mM Ca²⁺was2.45±1.67pA/pF at-100mV,42.25±14.09pA/pF at+100mV, and7.07±3.14pA/pF at+50mV. Characteristics of the endogenouscurrents in A549cell were significantly affected by extracellular Ca²⁺. First,the current density of inward current at-100mV, in the absence ofextracellular Ca²⁺was increased to23.54±9.06pA/pF, about10-folds of thatin the extracellular solution with1mM Ca²⁺. Second, the outward rectificationwas abolished by removing Ca²⁺from extracellular solution; the currentdensity in the Ca²⁺-free extracellular solution was41.38±16.93pA/pF at+100mV, which was not different compared with that in the extracellularsolution with1mM Ca²⁺(p>0.05); the outward current density was18.94±8.46pA/pF at+50mV in the Ca²⁺-free extracellular solution, which was about3-folds compared with that in the extracellular solution with1mM Ca²⁺. Thesecharacteristics of the whole-cell current in A549cells are similar to those ofTRPM7currents hence we refer these currents as TRPM7-like currents in thefollowing parts.2The mRNA and protein expression of TRPM7in A549cells and in tissue oflung adenocarcinomaThe gene and protein expression of TRPM7in A549cells was tested withRT-PCR and Western blot techniques. Abundant mRNA and membraneprotein expression of TRPM7were found in A549cells. Furthermore, we detected the protein expression of TRPM7in human lung adenocarcinomatissue by immunohistochemistry. The results suggested that TRPM7highlyexpressed not only in A549cell line, but also in native lung cancer tissues.The results supported the functional study that the current we recorded was theTRPM7current.32-APB inhibits the TRPM7-like current2-APB (a potent non-specific inhibitor of TRPM7channel) inhibited theoutward current of TRPM7-like current of A549cell recorded in theextracellular solution with1mM Ca²⁺. On average,2-APB (200μ M)decreased the amplitude of the outward currents at+100mV by about70%.We used five different concentrations (100μM,200μM,500μM,1mM and5mM)2-APB to study the concentration-dependency of2-APB. The resultsshowed that2-APB from100μM to1mM concentration-dependentlydecreased the amplitude of TRPM7-like current. However,5mM2-APB didnot inhibit TRPM7-like current. The current density of TRPM7-like current inthe control extracellular solution was43.60±6.21, which was reduced to23.10±5.26,16.96±3.00,7.51±2.90and6.42±2.90by100μM,200μM,500μM and1mM2-APB,respectively. The effects of2-APB were readilyreversible upon washout.2-APB also inhibited the TRPM7-like inward currentin Ca²⁺-free extracellular solution.2-APB (200μM) inhibited the inwardcurrent at100mV by about90%. Interestingly,2-APB restored the propertyof outward rectification of TRPM7-like currents in the absence of extracellularCa²⁺.4Divalent cations in extracellular solution inhibit the TRPM7-like currents.The characteristics of TRPM7-like currents in A549cells were furtherinvestigated by studying the effects of extracellular divalent cations on thesecurrents. It seems that the TRPM7currents in A549cells had differentsensitivity to external Ca²⁺and Mg2+. Although both1mM Mg2+or1mMCa²⁺completely blocked the inward currents, they affected the outwardcurrents quite differently, with Ca²⁺showing much lower potency than Mg2+.1mM Mg2+decreased the outward current of TRPM7by about60%, while1 mM Ca²⁺had no effect. However, it should be noted that the divalentcations-induced inhibition of the TRPM7currents was voltage dependent; it isclear that both Ca²⁺and Mg2+inhibited the outward currents significantly atpotentials less than+100mV. At higher concentrations, both Mg2+and Ca²⁺inhibited the outward currents of TRPM7; at20mM, Ca²⁺inhibited theoutward currents recorded at+100mV to a similar extent as5mM Mg2+did;the outward current density of TRPM7-like current was39.28±6.02pA/pF,15.19±4.41pA/pF and15.56±3.96pA/pF at+100mV in the extracellularsolution with1mM Ca²⁺,20mM Ca²⁺and5mM Mg2+, respectively.Interestingly, both Ca²⁺and Mg2+of extracellular solution restored theproperty of outward rectification of the TRPM7-like currents in the absence ofextracellular Ca²⁺as other inhibitor2-APB did.5TRPM7encodes most of the recorded currents in A549cellsWe designed two shRNA for TRPM7(TRPM7-shRNA1, TRPM7-shRNA2) depress the expression of TRPM7in A549cells. We first tested theefficacy of these two shRNA in depressing TRPM7in A549cell. RT-PCRresults suggested that TRPM7-shRNA1was more efficient thanTRPM7-shRNA2. Thus we used shRNA1in the following experiments. Toget a higher efficiency of transfection, we constructed lentiviral particle ofTRPM7-shRNA1and used it for all following experiment. LenivirusTRPM7-shRNA1induced a significant suppression of TRPM7mRNA andprotein levels in A549cells72h after the transfection, in comparison with thecells transfected with the control negative-shRNA. In TRPM7-shRNAtransfected A549cells, the outward TRPM7current amplitude (at+100mV)was significantly reduced by about80%compared with that in thenegative-shRNA transfected A549cells (39.84±9.02pA/pF). In Mg2+-andCa²⁺-free extracellular solution, the inward currents recorded at100mVwere also reduced by about80%in TRPM7-shRNA transfected cellscompared with the cells transfected with negative-shRNA48–72h aftertransfection. All these results indicated that TRPM7encodes most of therecorded outward and inward currents in A549cells. Conclusion:(1) TRPM7is functionally expressed in A549cells.(2)TRPM7encodes most of the recorded outward and inward currents in A549cells.(3) Divalent cations of extracellular solution inhibit the TRPM7currentof A549cell with different efficiency.Part2EGF enhances the migration of cancer cells by up-regulation ofTRPM7Objective: To investigate the role of TRPM7in EGF-meddiatedmigration of A549cells.Methods: RT-PCR,Western blot, immunocytochemistry and confocalmicroscopy were used to investigate the mRNA and protein expression ofTRPM7in A549cells. Whole-cell patch-clamp technique was used to monitorthe effect of EGF on TRPM7current in A549cell. To record the currents, thecells were held at0mV and stimulated with voltage ramps from120to+120mV. Wound-healing and transwell technologies were used to evaluate themigration of cancer cells.Results:1EGF enhances the function and the membrane protein expression of TRPM7in A549cells.When A549cells were exposed to EGF (100ng/ml) for48h, theTRPM7currents were increased significantly. The outward currents (at+100mV and+50mV) were increased about2-folds. The selective inhibitor of EGFreceptor, AG1478(10μM) not only abolished the EGF-enhanced TRPM7currents but also reduced the basal TRPM7currents. EGF treatment did notincrease the inward currents (at100mV) in the presence of external1mMCa²⁺. In the absence of external Ca²⁺, EGF increased the amplitude of both theoutward and the inward currents about2-folds, which were both inhibited by2-APB.2-APB restored the outward rectification property of the TRPM7currents in the absence of external Ca²⁺treated with EGF. EGF did not affectthe TRPM7currents when it was incubated with A549cells less than24h.The expression of mRNA and membrane protein of the TRPM7in A549cellswas also up-regulated by treatment with EGF for48h. 2EGF enhances the migration of A549cells by up-regulation of TRPM7The A549cells incubated in RPMI1640medium (supplemented with10%fetal bovine serum) with or without EGF (100ng/ml) for48h were usedin the experiments. In the wound-healing assays, the area that the cells hadmigrated within16h, toward initially scratched middle line from the borderline was measured. The cells stimulated with100ng/ml EGF migrated acrossan area that was2-folds as large as that of the cells incubated with the controlmedium. The effects of EGF on cell migration were inhibited by200μM2-APB and two inhibitors of EGF receptor, AG1478and genistein (10μM),the latter being a non-specific inhibitor of tyrosine kinase. AG1478andgenistein not only reversed EGF-induced enhancement of the migration butalso inhibited the control migration of A549cells, possibly due to a blockingeffect on activation of EGF pathway by the control medium.To further investigate the role of TRPM7in the migration of A549cells,we tested the effect of RNA interference for TRPM7. The wound-healingassay showed that TRPM7-shRNA1inhibited the migration area of A549cellsinduced by10%serum by about70%compared with the negative-shRNA.Furthermore, TRPM7-shRNA1also significantly reduced the migration areaof A549cells induced by EGF. The transwell assay gave similar results; EGFsignificantly increased the numbers of the migrating cells by about4-fold, andthe TRPM7-shRNA1not only reduced the cell migration induced by10%serum but also reduced the cell migration induced by EGF compared with thenegative-shRNA.Conclusion:(1) EGF enhances the function and the membrane proteinexpression of TRPM7in A549cells.(2) EGF enhances the migration of A549cells by up-regulation of TRPM7.Part3The molecular mechanisms for TRPM7involvement inEGF-induced migration of lung adenocarcinoma cellsObjective: Based on the experiment results that TRPM7was involved inthe EGF-induced migration of A549cells, we tried to study in this part themolecular mechanism for TRPM7involvement in the effect of EGF. Methods: Western blot was used to detect the protein expression of Aktand vimentin. Calcium photometry was used to measure the changes ofintracellular Ca²⁺concentration. Whole cell patch clamp technique was used tostudy the effect of inhibitor of PI3K and Akt on TRPM7currents. rhodamineor FITC-conjugated phalloidin were used to stain F-actin using confocalmicroscopy.Results:1PI3K-Akt signaling pathway is possibly involved in the EGF-inducedup-regulation of TRPM7current.Results of Western blot showed that EGF increased significantly theprotein expression of phosphorylated Akt (p-Akt) in A549cells stimulatedwith EGF for48hours, suggesting that EGF may up-regulate TRPM7throughAkt signaling pathway. To support this, although triciribine (Akt inhibitor) didnot inhibit the outward current of TRPM7, wortmanin (PI3K inhibitor) diddecrease the EGF-induced up-regulation of TRPM7.2EMT and the assembly of F-actin are also important for EGF-inducedmigration of A549cells and regulation of TRPM7Some morphology changes appeared in A549cells when treated withEGF (100ng/ml) for48h: the cells became smaller and the shape of the cellschanged from a cobblestone-like shape to a fibroblast-like appearance, and thenumber of single cells detached from the colonies was increased. The cellswith these typical changes were seen most often at the migration front. Thesecharacteristic changes are reminiscent of EMT (epithelial-mesenchymaltransition), which was proposed as the critical mechanism for the acquisitionof malignant phenotypes of epithelial cancer cells. Furthermore, the result ofWestern blot showed that EGF increased the protein expression of vimentin(typical protein of EMT). These results indicate that EGF induced EMT inA549cell, and EMT possibly played important roles in EGF-indcuedmigration of A549cell.Cellular motility was driven by assembly and disassembly of actinfilaments. To visualize the effect of EGF on the actin cytoskeleton, we used FITC or rhodamin-conjugated phalloidin, which specifically binds to thefilamentous actin (F-actin). EGF significantly increased the density of F-actinand induced the reorganization of the actin cytoskeleton from long to shortactin filaments. TRPM7-shRNA inhibited the effects of EGF on reorganizationof actin filaments.3TRPM7is responsible for Ca²⁺entry in A549cellUsing ratio Ca²⁺imaging measurements, we found that Ca²⁺influxinduced by high external Ca²⁺in A549cell was decreased after treated withTRPM7-shRNA1for3days, indicating that TRPM7is the major Ca²⁺permeable channel in this lung cancer cell, and the Ca²⁺influx throughTRPM7is possibly important for the effect of EGF on the migration of cancercells.Conclusion:(1) PI3K-Akt signaling pathway is possibly involved in theup-regulation of TRPM7protein expression and TRPM7current in A549cellinduced by EGF.(2) EMT and assembly of F-actin could be importantmechanisms for TRPM7involvement in the migration of A549cells inducedby EGF.(3) TRPM7is the major Ca²⁺permeable channel in this A549cell,which is possibly important for the effect of EGF on the migration of cancercells.